(A) γ-TuRCs were attached and 7μM tubulin (pseudo-colored in red) ± 20nM XMAP215-GFP (pseudo-colored in green) was added. Scale bar, 10 μm. Experiments and analyses in (A–B) were repeated thrice with independent γ-TuRC preparations. (B) Number of MTs nucleated (N(t)) over time (t) was measured and control reactions at 120 s was set to 1 to account for variable γ-TuRC concentration across purifications, all data were pooled and reported. Individual datasets with ±XMAP215 is represented with solid or dashed curves. Shaded regions represent 95% confidence interval from each dataset in the number of nucleated MTs assuming a Poisson distribution as described in Materials and methods. See also Figure 7—figure supplement 1A-B. (C) Sequence of events during cooperative MT nucleation by γ-TuRC and XMAP215 was visualized using labeled γ-TuRC (blue), XMAP215 (red) and tubulin (green) represented in a time sequence and kymograph. γ-TuRC and XMAP215 form a complex prior to MT nucleation. XMAP215 molecules reside on γ-TuRC for before MT nucleation. The experiment was repeated a total of eight times with two independent γ-TuRC preparations and independent XMAP215 purifications. Scale bar, 5μm. (D–E) Number of MTs nucleated (N(t)) over time (t) was measured after titrating tubulin with constant γ-TuRC and XMAP215 concentration. XMAP215/γ-TuRC molecules nucleate MTs from 1.6 μM tubulin. Shaded regions represent 95% confidence interval in the number of nucleated MTs assuming a Poisson distribution as described in Materials and methods. (E) Number of tubulin dimers (n) in the critical nucleus on cooperative nucleation by γ-TuRC/XMAP215 was obtained as 3.3 ± 0.8 from the equation displayed on a log-log axis as detailed in Materials and methods. The rate of nucleation at 3.5μM was set to 1 to normalize differences in γ-TuRC concentration from individual experiments. Experiment and analyses in (D–E) was repeated thrice over the entire concentration range with independent γ-TuRC preparations, and fewer concentration points were repeated another two times. All five datasets were pooled and data points from a total of 18 nucleation-time curves are reported in (E). Simulations were adapted to understand how XMAP215 changes the thermodynamics of γ-TuRC-mediated nucleation. Parameter values used: , , , and . Compared to simulations for γ-TuRC alone (Figure S6A), either was increased 1.2-fold, as proposed previously (VanBuren et al., 2002), or both and were increased 1.2-fold. 200 simulations each were performed for a range of tubulin concentration 1.6-7 μM. Probability of MT nucleation (p(t)) versus time (t) is displayed in (D). The initial rate of nucleation was measured at each tubulin concentration and plotted against concentration on a log-log axis in (E). Linear curve was fit for n=5 simulated data points, and critical nucleus of 3.8 ± 0.3 αβ-tubulins. Increasing all longitudinal bond energies reproduces the effect of XMAP215 on γ-TuRC-mediated nucleation. (F) Number of MTs nucleated was measured to assess the effect of inhibitory MAPs MCAK or Stathmin on γ-TuRC-mediated nucleation. 10.5μM tubulin ± 10nM MCAK, or 7-10.5μM tubulin ± 2-5μM Stathmin was added to attached γ-TuRC- molecules, and MCAK and Stathmin were both found to inhibit γ-TuRC-mediated nucleation. Experiments and analyses for both MAPs were repeated thrice individually with independent γ-TuRC preparations. Number of MTs nucleated in control reactions at 200 seconds was set to 1 to account for variable γ-TuRC concentration across purifications, all data was pooled and reported. Individual dataset with ± MCAK are reported with solid, dashed or dotted curves. For Stathmin, two datasets for 10.5 μM tubulin ± 5 μM Stathmin are reported with solid and dashed lines, and one dataset for 7 μM tubulin ± 2 μM Stathmin in dotted line. Shaded regions represent 95% confidence interval from each dataset in the number of nucleated MTs assuming a Poisson distribution as described in Materials and methods. See Figure 7—figre supplement 1, 2 and Videos 7, 8, 9.