Fig. 3.

Extent of MSA accumulation at precatastrophe microtubule tips depends on the match between the seed and growth conditions. (A and B) Representative kymographs illustrating drug accumulations in mismatching conditions. Microtubules were grown from GMPCPP seeds (A) or Taxol seeds (B) in the presence of 15 μM tubulin and 20 nM mCherry-EB3 with Fchitax-3 (100 nM) (A) or Alexa₄₈₈-epothilone B (100 nM) (B). White arrows indicate stable rescue sites with drug accumulations. (C and D) Representative kymographs illustrating drug accumulations in matching conditions. Microtubules were grown from GMPCPP seeds (C) or Fchitax-3 seeds (D) in the presence of 15 μM tubulin and 20 nM mCherry-EB3 with Alexa₄₈₈-epothilone B (100 nM) (C) or Fchitax-3 (100 nM) (D). Split comets are indicated with white arrows. (E) Quantification of drug accumulation lengths in the indicated conditions. Error bars represent SD; from left to right, n = 39, 34, 30, and 50; n = 3 independent experiments; ***P < 0.001. (F) Time plot of averaged normalized maximum intensity of fitted EB3 comet (orange and red) and normalized area under the curve (AUC) of fitted Fchitax-3 (light and dark green) intensity profiles in mismatching conditions (as shown in A; n = 9 kymographs from five experiments) and in matching conditions (as shown in D; n = 38 kymographs from five experiments). Individual curves were aligned by maximizing cross-correlation between Fchitax-3 time curves. Error bars represent SEM. (G) Frequencies of catastrophes (calculated as the frequency of all growth perturbations including split comet events) and split comet events in different matching conditions. From left to right, n = 65, 100, 91, 71, and 70 for catastrophe frequencies for the indicated conditions and n = 60, 66, 73, 62, and 63 catching-up events from 30 microtubules for split comet frequencies; n = 3 independent experiments. Error bars represent SD.