Microtubules are highly dynamic cytoskeletal filaments essential for a wide range of cellular processes. Recent studies have shown that microtubule dynamics occur not only at their growing and shrinking ends but also within the lattice itself. Lattice damage, which arises from the dissociation of tubulin subunits, can be repaired by the incorporation of new subunits, thereby restoring microtubule integrity. However, precisely identifying sites of microtubule damage and repair has remained challenging. Here, using cell-based and in vitro reconstitution assays, we demonstrate that the microtubule-binding domain of the minus-end stabilizing protein CAMSAP3 CAM3(MBD) forms discrete islands along microtubules, regardless of tubulin conformation or nucleotide state within the lattice. These islands preferentially localize at microtubule ends, crossings, and bundled regions, which were previously implicated as sites of mechanical stress-induced damage. Upon inducing microtubule lattice defects with motor proteins and external physical stress in cells and in vitro, we observed a significant increase in the density of CAM3(MBD) islands. Further analysis reveals that CAM3(MBD) binds to microtubule damage sites by recognizing incomplete microtubule protofilaments. Finally, we show that CAM3(MBD) can track real-time changes at damage sites as microtubule lattices become defective. Overall, our findings reveal that CAM3(MBD) specifically binds to microtubule damage sites, providing a potential molecular probe for investigating microtubule dynamics within the lattice.
Yue et al. (Sun,) studied this question.