Figure 2. FAP20-mediated recruitment of tubulin to the microtubule lattice.

(A) Representative TIRF images of GTP microtubules grown from GMPCPP seeds (magenta) in the presence of 10 μM tubulin (cyan) with and without 5 μM FAP20 at different time points, as indicated.

(B) Representative TIRF images of GMPCPP seeds (magenta) in the presence of 10 μM tubulin (cyan) with and without 5 μM FAP20 in the absence of GTP at different time points, as indicated.

(C) Mean fluorescence intensity of Cy5-labelled free tubulin along the GMPCPP seed with time for 10 min in the presence of 10 μM tubulin (light blue), +GTP (pink), +5 μM FAP20 (dark blue), and +5 μM FAP20 with GTP (red). Error bars represent the standard error of mean for values combined from three independent experiments.

(D) Fluorescence intensity line scans of Cy5-labelled free tubulin along the length of a microtubule in the presence of 10 μM tubulin (pink) and GTP, + 5 μM FAP20 without GTP (dark blue), +0.5 μM FAP20 with GTP (red, dashed line), + 5 μM FAP20 with GTP (red) at the end of 10 min. The vertical black lines in each curve represent the ends of the GMPCPP seeds for the respective experiments.

(E) Differences in final (T = 10 min) and initial (T = 0 min) normalized fluorescence intensity of Cy5-labelled free tubulin recruited per length (μm) of GMPCPP seed are shown in presence of 10 μM tubulin (light blue), +GTP (pink), +5 μM FAP20 (dark blue), and +5 μM FAP20 with GTP (red). Values from each experimental set are represented as different symbols and error bars represent the standard error of mean for values combined from three independent experiments (N = 5 for each experiment).