Supplementary Figure 6: C-terminus of XMAP215 is required for microtubule nucleation with γ-TuRC, Related to Fig. 6.

(A) Related to Fig. 6a. In vitro MT polymerization assay performed with several XMAP215 constructs: wild-type XMAP215, TOG1-5, TOG12-Kloop and TOG5-CT. Representative kymographs for each construct are displayed showing 15 minutes of growth for a representative MT in the field of view. Scale bar, 5 μm. Each experiment was repeated at least twice with freshly purified proteins for TOG1-5, TOG5-CT and TOG12-Kloop proteins. No difference in growth speed was observed using either fresh or frozen wild-type XMAP215, and reactions were performed once each with freshly purified protein and frozen-thawed wild-type protein. (B) Related to Fig. 6b. Branching MT nucleation activity of XMAP215 constructs was observed in Xenopus egg extracts. All proteins were added back to XMAP215-depleted extracts at varying final concentration in the presence of 5.5 μM RanQ69L and 1 μM GST-TPX2 α3-7. Representative images for each construct are displayed at 540 seconds of the reaction. A representative plot displays the nucleation curves generated with varying concentrations of TOG1-5 protein and wild-type XMAP215 in one Xenopus egg extracts. Scale bar, 10 μm. Number of times the experiments were repeated with independent extract preparations: wild-type XMAP215 (8), TOG1-5 (4), TOG12-Kloop (3), TOG5-CT (2). See Supplementary Video 9. (C) Related to Fig. 6c, d. Purified γ-TuRC was incubated with wild-type XMAP215 or TOG1-5 to promote MT nucleation in vitro. Individual MTs observed in the field of view were counted. TOG1-5 shows much lower synergistic MT generation than wild-type XMAP215 with purified γ-TuRC, although at the highest concentration of TOG1-5 tested (120 nM), the number of MTs approaches that seen with 30 nM full-length XMAP215. Roughly 20 fields of view for each reaction condition were analyzed, and data from two independent experiments performed with fresh γ-TuRC preparations was pooled. The number of fields analysed (n): γ-TuRC (n = 62); XMAP215 30 nM (n = 43); γ-TuRC + XMAP215 30nM (n = 41); TOG1-5 30 nM (n = 20); TOG1-5 60 nM (n = 39); TOG1-5 120 nM (n = 44); γ-TuRC + TOG1-5 30 nM (n = 20); γ-TuRC + TOG1-5 60 nM (n = 41); γ-TuRC + TOG1-5 120 nM (n = 41). Mean ± s.d. is plotted. See Supplementary Table 2 for source data.