Figure 1: Gatastatin is a γ-tubulin-specific inhibitor.

(a) The chemical structure of gatastatin. (b) The effect of gatastatin on MT dynamics in vitro was monitored as described in the Methods section. Kymographs of Alexa647-labelled MT polymerization (red) from tetramethylrhodamine- and biotin-labelled GMPCPP MT seeds (green) in the presence of GTP and either 1% DMSO, 30 μM gatastatin or 30 μM AG1. Horizontal scale bar, 2 μm; vertical scale bar, 1 min. (c) Quantification of the compound’s impact on the velocity of MT growth. Data are average velocities±s.e.m. calculated from 31 MTs (plus end) and 28 MTs (minus end) for DMSO, 23 MTs (plus end) and 22 MTs (minus end) for gatastatin, 23 MTs (plus end) and 20 MTs (minus end) for AG1. One-way ANOVA with Tukey’s multiple comparisons test was used to determine the significance of the difference using the GraphPad Prizm 6 software. *P<0.0001. There is no significant difference between the DMSO and gatastatin samples (P>0.05). (d) The effect of gatastatin on the binding activity of γ-TuSC to paclitaxel-stabilized MTs. γ-TuSC was treated with 50 μM of gatastatin and was incubated with paclitaxel-stabilized MTs. MTs and γ-TuSC were visualized by immunofluorescence on the coverslips using anti-α-tubulin (green) and anti-His tag (red, γ-TuSC) antibodies, respectively. At least 440 MTs were counted in each experiment. Three independent experiments were performed. Error bars represent s.d. Two-tailed, paired Student’s t-test was used to obtain P value. (e) The effects of gatastatin on the RanQ69L- and DMSO-stimulated aster formation. Egg extracts with Cy3-tubulin and gatastatin were incubated for 20 min at 20 °C in the presence of RanQ69L or 5% DMSO. Aster formation was analysed by fluorescence microscopy. Scale bar, 5 μm. (f) The average light intensity of asters in at least 10 randomly selected fields with a × 10 objective was quantified. Three independent experiments were performed. Error bars represent s.d.