Figure 9.
The TPX2–Aurora A–WDR62–katanin module can potently disassemble GDP-MTs in vitro.(A–C) Images and quantification of 30 nM, 60 nM, and 120 nM p60/SNAP-p80 binding to preassembled dynamic MTs in flow-in experiments. At 60 nM, p60/p80 was also observed to bind and bend the MT end (B). n = 19–22 MTs from two experiments. (D) Time-lapse images showing no severing, a few severing events on GMPCPP seed (arrows), and efficient severing of preassembled dynamic MTs in flow-in experiments with 30 nM, 60 nM, and 120 nM p60/SNAP-p80, respectively. (E) Severing frequency of p60/p80 on GMPCPP seeds and GDP MTs at indicated concentrations. n = 2 experiments; 19–22 MTs were measured. (F) Images showing the recruitment of 30 nM p60/SNAP-p80 to dynamic MTs preassembled in the presence of 30 nM WDR62-GFP and indicated concentration of TPX2/TagBFP–Aurora A (AurA) in flow-in experiments. (H–J) Quantification of intensities of TPX2/Aurora A (H), WDR62 (I), and p60/p80 (J) on GMPCPP seeds and GDP MTs immediately after flow-in as shown in F. Values were normalized to the intensity in the condition with 30 nM TPX2/TagBFP–Aurora A, 30 nM WDR62-GFP, and 30 nM p60/SNAP-p80 on GMPCPP seeds. n = 20–30 MTs from two experiments. (G and K) Images and quantification of MT-severing frequency of 30 nM p60/SNAP-p80 in the presence of 30 nM WDR62-GFP and indicated concentration of TPX2/TagBFP–Aurora A. n = 2 experiments; 20–30 MTs were measured. (L and N) Images and quantification of intensities of p60/p80 on GMPCPP seeds and GDP MTs in the presence of indicated factors. All proteins were used at 30 nM. The "x" in L indicates the absence of TPX2/AurA or WDR62 in the corresponding assays. Values were normalized to the intensity in the condition with 30 nM TPX2/TagBFP–Aurora A, 30 nM WDR62-GFP, and 30 nM p60/SNAP-p80 on GMPCPP seeds. n = 20–30 MTs from two experiments. (M and O) Images and quantification of MT-severing frequency of p60/p80 on GMPCPP seeds and GDP MTs in the presence of indicated factors at the same concentrations as in L. Arrows indicate severing events on GMPCPP seeds. Of note, the severing frequency of p60/p80 on seeds in the presence of WDR62 and TPX2 was lower than that in the presence of WDR62 alone or WDR62 and Aurora A, which reflects the stabilization effect exerted by TPX2. n = 2 experiments; 20–30 MTs were measured. Scale bars, 2 µm. Data represent mean ± SD. N.D., not detected.

The TPX2–Aurora A–WDR62–katanin module can potently disassemble GDP-MTs in vitro.(A–C) Images and quantification of 30 nM, 60 nM, and 120 nM p60/SNAP-p80 binding to preassembled dynamic MTs in flow-in experiments. At 60 nM, p60/p80 was also observed to bind and bend the MT end (B). n = 19–22 MTs from two experiments. (D) Time-lapse images showing no severing, a few severing events on GMPCPP seed (arrows), and efficient severing of preassembled dynamic MTs in flow-in experiments with 30 nM, 60 nM, and 120 nM p60/SNAP-p80, respectively. (E) Severing frequency of p60/p80 on GMPCPP seeds and GDP MTs at indicated concentrations. n = 2 experiments; 19–22 MTs were measured. (F) Images showing the recruitment of 30 nM p60/SNAP-p80 to dynamic MTs preassembled in the presence of 30 nM WDR62-GFP and indicated concentration of TPX2/TagBFP–Aurora A (AurA) in flow-in experiments. (H–J) Quantification of intensities of TPX2/Aurora A (H), WDR62 (I), and p60/p80 (J) on GMPCPP seeds and GDP MTs immediately after flow-in as shown in F. Values were normalized to the intensity in the condition with 30 nM TPX2/TagBFP–Aurora A, 30 nM WDR62-GFP, and 30 nM p60/SNAP-p80 on GMPCPP seeds. n = 20–30 MTs from two experiments. (G and K) Images and quantification of MT-severing frequency of 30 nM p60/SNAP-p80 in the presence of 30 nM WDR62-GFP and indicated concentration of TPX2/TagBFP–Aurora A. n = 2 experiments; 20–30 MTs were measured. (L and N) Images and quantification of intensities of p60/p80 on GMPCPP seeds and GDP MTs in the presence of indicated factors. All proteins were used at 30 nM. The "x" in L indicates the absence of TPX2/AurA or WDR62 in the corresponding assays. Values were normalized to the intensity in the condition with 30 nM TPX2/TagBFP–Aurora A, 30 nM WDR62-GFP, and 30 nM p60/SNAP-p80 on GMPCPP seeds. n = 20–30 MTs from two experiments. (M and O) Images and quantification of MT-severing frequency of p60/p80 on GMPCPP seeds and GDP MTs in the presence of indicated factors at the same concentrations as in L. Arrows indicate severing events on GMPCPP seeds. Of note, the severing frequency of p60/p80 on seeds in the presence of WDR62 and TPX2 was lower than that in the presence of WDR62 alone or WDR62 and Aurora A, which reflects the stabilization effect exerted by TPX2. n = 2 experiments; 20–30 MTs were measured. Scale bars, 2 µm. Data represent mean ± SD. N.D., not detected.

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