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. 2020 Sep 15;31(20):2187–2194. doi: 10.1091/mbc.E20-02-0160

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© 2020 King et al. “ASCB®,” “The American Society for Cell Biology®,” and “Molecular Biology of the Cell®” are registered trademarks of The American Society for Cell Biology.

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FIGURE 3: — The microtubule nucleation activity of XMAP215 correlates strongly with its polymerase activity. (A) XMAP215 and XMAP215 deletion constructs used in this work. The red box represents the basic microtubule binding domain. An SDS–polyacrylamide gel showing the deletion constructs is given in Supplemental Figure 2. (B) Schematic of the assay used to measure tip growth rates from immobilized GMPCPP-stabilized microtubule seeds by microscopy. (C) Microtubule elongation rates at 12 μM free αβ-tubulin, with full-length XMAP215 or the indicated deletion constructs added at 40 nM, or with γ-tubulin added at 300 nM. Error bars represent SD (N = 41, 35, 33, 41, 49, 53, 41 microtubules measured for each respective condition). The asterisk represents p < 0.0001, two-sided t test. (D) Schematic of the assay used to measure nontemplated tubulin assembly by turbidity. (E) Representative turbidity assay at 15 μM free αβ-tubulin, with XMAP215 or the indicated deletion constructs added at 40 nM. The red dashed line represents 10% of the maximum light scattering. The inset shows the data indicated by the red box replotted on a larger scale with the data plotted as points connected by lines for clarity. (F) Nucleation activity plotted against polymerase activity. Data for XMAP215 and its deletion constructs, each added at 40 nM, are fit by a power curve with the equation (see Materials and Methods). Normalized microtubule growth rate means ± SEM represent the data from Figure 3C normalized to the growth rate of tubulin alone. Relative nucleation activity for each construct is represented by a delay between the start of the polymerization reaction and when the turbidity reached 10% of its maximum, divided by the delay measured in controls with αβ-tubulin alone. Normalized nucleation rate means ± SEM represent the continuous turbidity reaction data of experimental conditions normalized to the nucleation lag of tubulin alone (N = 12, 7, 6, 4, 6, 5, and 7 for tubulin alone, γ-tubulin, TOG12, TOG12MTbd, TOG1-4, TOG1-4MTbd, and XMAP215, respectively).

Inline graphic — The microtubule nucleation activity of XMAP215 correlates strongly with its polymerase activity. (A) XMAP215 and XMAP215 deletion constructs used in this work. The red box represents the basic microtubule binding domain. An SDS–polyacrylamide gel showing the deletion constructs is given in Supplemental Figure 2. (B) Schematic of the assay used to measure tip growth rates from immobilized GMPCPP-stabilized microtubule seeds by microscopy. (C) Microtubule elongation rates at 12 μM free αβ-tubulin, with full-length XMAP215 or the indicated deletion constructs added at 40 nM, or with γ-tubulin added at 300 nM. Error bars represent SD (N = 41, 35, 33, 41, 49, 53, 41 microtubules measured for each respective condition). The asterisk represents p < 0.0001, two-sided t test. (D) Schematic of the assay used to measure nontemplated tubulin assembly by turbidity. (E) Representative turbidity assay at 15 μM free αβ-tubulin, with XMAP215 or the indicated deletion constructs added at 40 nM. The red dashed line represents 10% of the maximum light scattering. The inset shows the data indicated by the red box replotted on a larger scale with the data plotted as points connected by lines for clarity. (F) Nucleation activity plotted against polymerase activity. Data for XMAP215 and its deletion constructs, each added at 40 nM, are fit by a power curve with the equation (see Materials and Methods). Normalized microtubule growth rate means ± SEM represent the data from Figure 3C normalized to the growth rate of tubulin alone. Relative nucleation activity for each construct is represented by a delay between the start of the polymerization reaction and when the turbidity reached 10% of its maximum, divided by the delay measured in controls with αβ-tubulin alone. Normalized nucleation rate means ± SEM represent the continuous turbidity reaction data of experimental conditions normalized to the nucleation lag of tubulin alone (N = 12, 7, 6, 4, 6, 5, and 7 for tubulin alone, γ-tubulin, TOG12, TOG12MTbd, TOG1-4, TOG1-4MTbd, and XMAP215, respectively).