Data for: The conserved centrosomin motif, γTuNA, forms a dimer that directly activates microtubule nucleation by the γ-tubulin ring complex (γTuRC)

To establish the microtubule cytoskeleton, the cell must tightly regulate when and where microtubules are nucleated. This regulation involves controlling the initial nucleation template, the γ-tubulin ring complex (γTuRC). Although γTuRC is present throughout the cytoplasm, its activity is restricted to specific sites including the centrosome and Golgi. The well-conserved γ-tubulin nucleation activator (γTuNA) domain has been reported to increase the number of microtubules (MTs) generated by γTuRCs. However, previously we and others observed that γTuNA had a minimal effect on the activity of antibody-purified Xenopus γTuRCs in vitro (Thawani et al., eLife, 2020; Liu et al., 2020). Here we instead report, based on improved versions of γTuRC, γTuNA, and our TIRF assay, the first real-time observation that γTuNA directly increases γTuRC activity in vitro, which is thus a bona fide γTuRC activator. We further validate this effect in Xenopus egg extract. Via mutation analysis, we find that γ..., Short description:
The archive contains the following: 
1) raw microscopy images, videos collected via total internal reflection fluorescence (TIRF) for the following:
    1a) Xenopus laevis egg extracts treated with gTuNA (with fluorescent end-binding protein 1 marking growing microtubule plus-ends);
    1b) in vitro nucleation assays with purified gTuRC and gTuNA at 15 µM tubulin (with fluorescent tubulin to visualize microtubules);
    1c) in vitro assays with purified gTuNA bound to beads (post-extract pulldown);
    1d) in vitro nucleation assays with purified gTuRC and gTuNA at 7 µM tubulin (critical concentration for gTuRC activity);
    1e) in vitro nucleation assays with purified gTuRC, gTuNA, and stathmin at 15 µM tubulin;
2) images of purified gTuRCs generated via negative-stain electron microscopy;
3) size-exclusion chromatography files generated by GE Unicorn software (for Akta Pure Systems);
4) mass spectrometry data to identify co-precipitants of gTuNA from extract pulldo..., The microscopy data (either Nikon \".nd2\" or \".tif\" extensions) can be opened using the open-source program ImageJ/FIJI (with the BioFormats plugin installed and the Nikon \".nd2\" library enabled). This same program can open the \".ijm\" extension macros. CSV and TXT files can be opened with most text-based editors, although we used Microsoft Excel or MATLAB to view or process them. MATLAB code requires access to MathWorks MATLAB environment, although an open-source alternative, GNU-Octave, can be used. However, GNU-Octave does not contain all the required dependencies for all the scripts included here.