TPX2 α5–α7 is the minimal construct that stimulates branching MT nucleation. (A) Domain organization of the C-terminal half of X. laevis TPX2 based on secondary structure prediction using Jpred. (B) Graphical representation of the FKARP motif, which occurs after predicted α-helices in domains α3 through α6. (C) CD spectra of various TPX2 constructs in the region of 200–250 nm. All spectra suggest the presence of significant α-helical content, which is characterized by two minima near 208 nm and 220 nm. Estimates of secondary structure content using the K2D3 method agree with values obtained from the Jpred prediction. (D–M) Branching MT nucleation in Xenopus egg extracts in the presence of 2 µM of different truncated TPX2 constructs. The same results were obtained with a lower TPX2 concentration of 0.75 µM. EB1-GFP (green) and Alexa Fluor 568–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 gliding of MTs. All images were taken after 5 min. Brightness and contrast were adjusted for each image individually to optimize visual comparison of MT structures. Bar, 10 µm. See Video 1. (N) The number of individual MTs was counted for each time frame and then plotted against time. The data are displayed for addition of TPX2 α3–α7 and all the inactive constructs. (O) Same as N, except the number of MTs is averaged over two experimental replicates for the addition of TPX2 α3–α7 and the other active constructs α4–α7 and α5–α7. Error bars represent absolute error. All extract experiments were performed at least three different times.