Fig. 4: Biochemical characterization of mosaic 20%Q + N-microtubules.
From: Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap
A Plot of average maximum mGFP-EB3 fluorescence intensities measured along 20%Q + N-microtubules at different mGFP-EB3 concentrations. N = 2 independent experiments, n = 100 microtubules per mGFP-EB3 concentration. Error bars represent SEM. A hyperbolic fit yields the dissociation constant, Kd, as indicated. Fluorescence intensities are represented in arbitrary units (A.U.). B Plot of average microtubule growth rates at different total tubulin concentrations in the presence of 40 nM mGFP-EB3 and the respective linear regressions for αE254E (WT,circles, green line), αE254Q (triangles, blue line), αE254N (squares, orange line), and 20%Q + N-microtubules (diamonds, purple line). N = 3 independent experiments; a total of 29–362 growth events were analyzed per condition. Error bars represent SEM. From the slope and the intercept, we determined the apparent on-rate constant kon,app and off-rate constant koff,app, respectively, as summarized in Supplementary Table 1. C TIRF microscopy kymographs of αE254E (WT), αE254N, αE254Q, and mosaic 20%Q + N-microtubules for the 10 µM tubulin condition in (B), polymerized from stabilized seeds (purple) in the presence of 40 nM mGFP-EB3 (green). Horizontal and vertical scale bars represent 10 μm and 5 min, respectively. D Time course of the turbidity after start of polymerization of 20%Q + N-microtubules at different total tubulin concentrations as indicated. The inset shows a magnification of the beginning of the polymerization reaction. Black circles indicate where the absorbance reaches 10% of its maximum value (10% Amax) which is used to calculate the apparent self-nucleation rate constant. E Plots to determine the apparent self-nucleation rate constant K* of WT (circles, green linear regression), αE254Q (triangles-down, blue linear regression), and 20%Q + N-tubulin (triangles-up, purple linear regression). N = 3 independent experiments; n = 2 replicas per condition. Error bars represent SEM. F IRM images of microtubules spontaneously nucleated in solution from different tubulins, as indicated, at the concentrations, as indicated. Scale bars represent 10 µm. Source data are provided as a Source Data file.
