Fig. 5.

Microtubule severing at the stable rescue site suppresses catastrophes. (A and B) Schematic representation and still images showing photoablation of microtubule regions within (A) or before (B) a drug accumulation zone (SRS) and kymographs showing microtubule dynamics of the severed parts. The assay was performed in the presence of GMPCPP seeds with 15 μM tubulin supplemented with 3% rhodamine-tubulin, 20 nM mCherry-EB3, and 100 nM Fchitax-3. The time-lapse images before photoablation show the presence of stable rescue sites; the site of ablation within (A) and before (B) the stable rescue site is indicated by a lightning bolt. The newly generated microtubule fragments (1: seed-attached part; 2: the part that is detached from the seed after ablation), microtubule plus (+) ends, and the new plus (+) and minus (−) ends generated after ablation are indicated. In time-lapse images, # indicates the position of the GMPCPP seed. (A) The kymograph illustrates the dynamics of both fragments (1 and 2, as shown in the scheme) generated after ablation. (B) The kymograph illustrates the dynamics of the severed microtubule fragment (2, as shown in the scheme). Asterisks highlight catastrophes, and rescues at the stable rescue site are indicated by arrows in B. The labels are in blue for fragment 1 (seed-attached microtubule part) and yellow for fragment 2 (the part detached after ablation); n = 5 independent experiments. (C) Quantification of growth dynamics of the unsevered and severed microtubule segment in matching (n = 75, no severing; n = 35, after severing) and mismatching conditions; n = 100, 60, and 55 for no ablation, or ablation either before or within a drug accumulation area, respectively. (D) Quantification of catastrophe frequencies (calculated as the frequency of all growth perturbations including catching-up events). Matching conditions (Fchitax-3 seeds + 100 nM Fchitax-3): n = 100 (no ablation) and n = 35 (after ablation). Mismatching conditions (GMPCPP seeds + 100 nM Fchitax-3): n = 76 (no ablation, after the formation of a stable rescue site), n = 115 (after photoablation in a region preceding the Fchitax-3 accumulation area [SRS]), and n = 39 (after photoablation within SRS). ns, not significant; ****P < 0.0001, Mann–Whitney U test. (E) Model depicting microtubule lattice repair in matching and mismatching conditions. In the precatastrophe state, MSAs stabilize depolymerizing protofilaments and inhibit depolymerization. In matching conditions, this leads to rapid repair of microtubule defects and restoration of microtubule growth. In mismatching conditions, lattice defects persist despite repair due to a switch in protofilament number. The presence of a defect might generate strain in the lattice, which would affect the growing microtubule end and induce catastrophe.