Supplementary Figure 2: Microtubule polymerization in Xenopus egg extracts occurs with low XMAP215 concentration, Related to Fig. 2.

(A) De novo nucleation events were observed with increasing XMAP215 concentration (Fig. 2a, b). All EB1 comets in the field of view were tracked during the entire reaction, and growth speed of MTs was obtained from consecutive frames of all tracks. No MTs were observed when buffer (0 nM), 15 nM or 30 nM XMAP215 was added back; therefore, growth speed could not be measured. Mean growth speed for 60 nM and above XMAP215 concentrations is plotted. Error bars represent s.d. in all measurements. The number of growth speed measurements obtained from consecutive frames of all tracks (n). 60 nM (n = 87), 120 nM (n = 734), 240 nM (n = 1,889), 480 nM (n = 19,379), 720 nM (n = 43,995). Mean speed versus XMAP215 concentration was fitted to Michaelis-Menten kinetics (dashed curve). See Supplementary Video 3. The analysis was repeated twice with independent extract preparations. (B-C) Stabilized GMPCPP MTs (blue) were digested with subtilisin A and attached to passivated coverslips. Growth from MT seeds (red – tubulin, green – EB1-mCherry) was observed in XMAP215-depleted extracts supplemented with low XMAP215 concentration. Distinct MT growth events were observed starting from 7.5 nM XMAP215, while EB1 blinking events were seen at one end of seeds without any XMAP215 added. Scale bar, 10μm. Representative kymographs of these events are displayed in (C). This experiment was repeated thrice with independent extract preparations. (D) Growth speed was measured from kymograph analysis. All measurements versus XMAP215 concentration and fitted to Michaelis-Menten kinetics (red curve), and s.d. displayed in shaded red area. The number of kymographs analyzed (n): buffer (n = 10), 7.5 nM (n = 11), 15 nM (n = 16), 30 nM (n = 18), 60 nM (n = 17). The analysis was repeated on two independent experimental sets. (E) Centrally illuminated [104 × 104 μm] region was selected. All MT seeds were observed for a short period of 35–80 seconds. The number of MT seeds analyzed (n): buffer (n = 47), 7.5 nM (n = 100), additional reaction at 7.5 nM (n = 116), 15 nM (n = 31), 30 nM (n = 85), 60 nM (n = 65). Fraction of seeds that displayed MT growth on one end, based on composite tubulin and EB1 signal, was reported (blue diamonds). For buffer (0 nM XMAP215) addition, fraction of seeds displaying distinct EB1 blinking events on one end was reported (red square). The analysis was repeated on two independent experimental sets and one supporting set. See Supplementary Table 2 for source data.