Figure 3. Cytoskeleton-Associated Protein 2 (CKAP2) increases templated and spontaneous microtubule nucleation.
(a) Representative field of view of guanosine-5′-[(α,β)-methyleno]triphosphate (GMPCPP)-stabilized microtubule seeds nucleating microtubules from 8 µM tubulin. (b) Plot of the cumulative probability distributions for time to nucleate as a function of CKAP2-mNeonGreen (mNG) concentration (n = 67, 70, 67, 46 from two replicates). (c) Plot of probability of GMPCPP seed to be nucleated within 1 min for control (blue; n = 102, 271, 115, 184 from ≥2 replicates), 0.125 µM CKAP2-mNG (green; n = 164, 213, 166 from two replicates), 0.50 µM CKAP2-mNG (red; n = 179, 216, 145, 126, 145 from two replicates), and 1 µM CKAP2-mNG (purple; n = 112, 186, 182, 148 from two replicates). Plotted as mean ± standard deviation (SD). Data were fit to a hill function forced to start at y = 0 and end at y = 1 (y = START + (END − START) × xn/(kn + xn)). Tubulin concentrations for half-maximal microtubule growth, C, and steepness of fit, s, for control (C = 6.85 ± 0.48 μM, s = 3.37 ± 0.87), 0.125 µM CKAP2-mNG (C = 0.18 ± 0.01 μM, s = 6.96 ± 1.39), 0.5 µM CKAP2-mNG (C = 0.05 ± 0.01 μM, s = 3.10 ± 0.20), and 1 µM CKAP2-mNG (C = 0.05 ± 0.01 μM, s = 3.79 ± 0.85). (d) Enlargement at low tubulin concentrations of probability to nucleate plot from (c). (e) Representative field of view of microtubules spontaneously nucleated in the absence of templates. (f) Quantification of spontaneous nucleation and linear fit showing a critical tubulin concentration for microtubule nucleation of 25.4 ± 1.7 µM for tubulin alone (blue; n = 20) and 0.25 ± 0.02 µM in the presence of 0.2 µM CKAP2 and 0.25 ± 0.02 µM (red; n = 14). (g) Enlargement at low tubulin concentrations of spontaneous nucleation in the presence of 0.2 µM CKAP2 from (e).
Figure 3—figure supplement 1. Cytoskeleton-Associated Protein 2 (CKAP2) increases templated and spontaneous microtubule nucleation.
