Aumeier, C.; Egoldt, C.; Reymond, L.; Tran, J.; Salim, A.; Velluz, M.-C.; Hoogendoorn, S.

Microtubules experience mechanical and enzymatic stresses that can compromise lattice integrity, yet where and how lattice damage forms remains poorly understood due to the lack of tools that directly visualize damage as it occurs. Existing approaches infer damage indirectly through repair events or rely on static ultrastructural snapshots, precluding dynamic analysis. Here, we introduce MT-DS (Microtubule Damage Sensor), a damage-selective fluorescent probe that directly labels microtubule lattice openings. MT-DS combines taxane-based microtubule binding with a multivalent protein scaffold to restrict intraluminal diffusion and selectively retain the probe at sites of lattice openings. Using MT-DS, we visualize intrinsic lattice defects in stabilized microtubules, uncover a strong enrichment of damage at annealing sites, and demonstrate that kinesin-1{Delta}6 actively generates de novo lattice damage during motility. By enabling direct, time-resolved detection of microtubule damage, MT-DS establishes lattice integrity as an experimentally accessible parameter and provides a chemical tool to investigate how mechanical stress reshapes the microtubule cytoskeleton.