Branching MT nucleation activity of multiple TPX2 single-site mutants. (A) Domain organization of TPX2 α5–α7 showing the location of single-site mutations. (B–L) Branching MT nucleation in Xenopus egg meiotic extracts and in the presence of 2 µM TPX2 α5–α7 with and without various single-site mutations. The same results were obtained with a lower TPX2 concentration of 1 µM. Images for F492AzF, F629AzF, and F714AzF mutants, which show a high level of activity, as well as the image for the positive control TPX2 α5–α7, were collected using one extract. Images for the rest of the mutants and the negative control were collected with a different extract. This was necessary because the lifetime of one extract was incompatible with the high number of samples; however, both controls are representative and consistent among all extracts tested. EB1-mCherry (green) and Cy5-labeled porcine brain tubulin (red) were added to the extract to follow MT plus ends and MTs, respectively. Vanadate was added to prevent dynein-mediated MT gliding. All images were acquired after 15 min. Brightness and contrast were adjusted for each image individually to optimize visual comparison of MT structures. Bar, 10 µm. See Video 3. (M) The number of EB1 particles was counted for three different fields of view and the mean was plotted against time. The data are displayed for the addition of TPX2 α5–α7 and all the inactive mutants. Data collection for the positive control, TPX2 α5–α7, was interrupted in the region denoted by the dashed line. Error bars represent standard deviation. (N) Same as M, but the data are depicted for the addition of TPX2 α5–α7 and mutants that have full or intermediate activity. Measurements also represent the mean of three different fields of view, and the error bars denote standard deviation. All extract experiments were performed at least three different times.