Figure 4.
Individual contribution of distinct TPX2 repeat groups in vitro. (A) Images and corresponding kymographs showing the behavior of 10 nM WT or indicated mutant versions of GFP-TPX2 FL on dynamic MTs grown in our standard assay buffer containing 50 mM KCl. Arrows in images and kymographs indicate gaps along GDP-MT lattices. (B) Quantification of intensities of 10 nM WT or indicated mutant versions of GFP-TPX2 FL on GMPCPP seeds (left) and GDP-MT lattices (right). The values were normalized to the average intensity of GFP-TPX2 FL WT. N.D., not detected. n = 14–49 MTs from two experiments. (C) Quantification of the gap index in the presence of 10 nM WT or indicated mutant versions of GFP-TPX2 FL. The values were calculated by dividing the length of gaps by the length of the entire GDP-MT lattice and averaged over three time points (2, 4, and 8 min). n = 13–17 MTs from two experiments. (D) Quantification of the probability of MT nucleation from GMPCPP seeds at tubulin concentrations ranging from 2 to 8 µM in the presence of 100 nM WT or indicated mutant versions of GFP-TPX2 FL. Reaction in the absence of the TPX2 protein was used as a control. n = 3 experiments (98–224 MTs were measured for each condition). The horizontal dashed line indicates a 50% probability of MT nucleation. (E) Kymographs showing the behavior of 100 nM WT or indicated mutant versions of GFP-TPX2 FL at 4 µM tubulin. (F–I) Quantification of the MT growth rate (F), depolymerization rate (G), catastrophe frequency (H), and rescue frequency (I) in the presence of 100 nM WT or indicated mutant versions of GFP-TPX2 FL at 4 µM tubulin. n = 53–107 events from three experiments (F); n = 36–72 events from three experiments (G); n = 27–66 MTs from three experiments (H); n = 25–37 MTs from three experiments (I). (J) Quantification of the lag time between the end of a shrinking event and the initiation of the MT regrowth (indicated by the vertical dashed lines in E). n = 35–73 events from three experiments. Horizontal scale bars, 2 µm; vertical scale bars, 2 min. Data represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant, two-tailed t test (unpaired). Refer to the image caption for details.

Individual contribution of distinct TPX2 repeat groups in vitro. (A) Images and corresponding kymographs showing the behavior of 10 nM WT or indicated mutant versions of GFP-TPX2 FL on dynamic MTs grown in our standard assay buffer containing 50 mM KCl. Arrows in images and kymographs indicate gaps along GDP-MT lattices. (B) Quantification of intensities of 10 nM WT or indicated mutant versions of GFP-TPX2 FL on GMPCPP seeds (left) and GDP-MT lattices (right). The values were normalized to the average intensity of GFP-TPX2 FL WT. N.D., not detected. n = 14–49 MTs from two experiments. (C) Quantification of the gap index in the presence of 10 nM WT or indicated mutant versions of GFP-TPX2 FL. The values were calculated by dividing the length of gaps by the length of the entire GDP-MT lattice and averaged over three time points (2, 4, and 8 min). n = 13–17 MTs from two experiments. (D) Quantification of the probability of MT nucleation from GMPCPP seeds at tubulin concentrations ranging from 2 to 8 µM in the presence of 100 nM WT or indicated mutant versions of GFP-TPX2 FL. Reaction in the absence of the TPX2 protein was used as a control. n = 3 experiments (98–224 MTs were measured for each condition). The horizontal dashed line indicates a 50% probability of MT nucleation. (E) Kymographs showing the behavior of 100 nM WT or indicated mutant versions of GFP-TPX2 FL at 4 µM tubulin. (F–I) Quantification of the MT growth rate (F), depolymerization rate (G), catastrophe frequency (H), and rescue frequency (I) in the presence of 100 nM WT or indicated mutant versions of GFP-TPX2 FL at 4 µM tubulin. n = 53–107 events from three experiments (F); n = 36–72 events from three experiments (G); n = 27–66 MTs from three experiments (H); n = 25–37 MTs from three experiments (I). (J) Quantification of the lag time between the end of a shrinking event and the initiation of the MT regrowth (indicated by the vertical dashed lines in E). n = 35–73 events from three experiments. Horizontal scale bars, 2 µm; vertical scale bars, 2 min. Data represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant, two-tailed t test (unpaired).

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