Extended Data Fig. 1: TIRF-based optimization of γ-TuRC-dependent microtubule nucleation assay with slow GTP hydrolysing mutant of ɑ-tubulin, E254D.
From: Structure of the γ-tubulin ring complex-capped microtubule
(a) Transmission EM micrograph of negatively stained native γ-TuRC (representative image from 441 micrographs). (b) 2D class-averages showing two orientations of native γ-TuRC particles, top view (upper panel, 1182 particles) and side-view (bottom panel, 1048 particles) from negative stain EM. (c,d) 2D class averages for native γ-TuRC-capped minus-ends (177 ends, 125 from which were used for protofilament number analysis) (c) and for uncapped ends from spontaneously nucleated microtubules (183 ends) from cryo-EM (d). (e) SDS-PAGE analysis (Coomassie stained) of recombinant γ-TuRC-GFP following sucrose density gradient centrifugation. Expected positions for the components of the complex are indicated. (f,g) Images from a TIRF-microscopy sequence showing the γ-TuRC (green), tubulin (magenta) and merge channels after 2 minutes 30 s in the presence of chTOG (100 nM) and either wild type tubulin (10 µM) (f) or E254D (TUBA1B-E254D, TUBB3) tubulin (10 µM) (g). (h) Plot of the percentage (mean ± s.d.) of microtubules associated with a γ-TuRC-GFP puncta or not associated with a γ-TuRC-GFP puncta at 50 s post start of nucleation in the presence of E254D tubulin (10 µM) and chTOG (100 nM), total 95 microtubules, n = 3 independent experiments for each condition. (i) Plot of the cumulative number of microtubules (mean ± s.d.) nucleated by γ-TuRC in the presence of chTOG (100 nM) and either wild type tubulin or E254D tubulin (10 µM) over time, is shown. Data were fitted using linear regression, E254D tubulin (red line, total 193 microtubules) and wild type tubulin (blue line, no microtubules were detected). n = 4 independent experiments for each condition.
