TPX2 MT nucleation activity is regulated by a 7–amino acid sequence that contributes to spindle pole morphology but not length. (A) Domain schematic of the X. laevis TPX2 protein. The first 39 amino acids are unstructured and interact with Aurora A (orange; Bayliss et al., 2003) and the last 35 amino acids interact with Eg5 (green; Bayliss et al., 2003; Eckerdt et al., 2008). A mapped nuclear localization signal is at amino acid 284. Three highly conserved domains are denoted by dark purple shading (Goshima, 2011). Zoom-in shows conservation between X. laevis and human of a 7–amino acid sequence missing from the X. tropicalis orthologue. (B) Addition of 200 nM of recombinant TPX2 mutants to X. laevis extract. (top) 20× field of view showing increased MT aster structures nucleated upon addition of X. tropicalis TPX2 or X. laevis Δ7 TPX2 compared with control or X. laevis TPX2. Arrowheads indicate MT asters. (bottom) Spindle morphology after TPX2 mutant addition. X. tropicalis TPX2 and X. laevis Δ7 TPX2 induced formation of radial astral MTs emanating from the poles not seen in control spindles or with X. laevis TPX2 addition. Bars, 10 µm. (C) Quantification of nucleation activity of recombinant TPX2 proteins. For each condition, spindle reactions were sedimented onto coverslips as described in Materials and methods and the number of MT aster structures was counted in 10 microscope fields, repeated in three separate extracts. Boxplot of number of asters per field with median marked by gray line, first and third quartiles marked by box edges, and data maxima and minima noted by whiskers. (D) Quantification of pole-to-pole spindle length, not including astral MTs, with addition of 200 nM MBP-TPX2 proteins compared with control of addition of 200 nM MBP. Mean ± SD; n ≥ 671 spindles in each condition from three separate extracts. For each TPX2 protein compared with MBP control, P < 0.0001 from unpaired t test.