The TPX2–Aurora A interaction is required for spindle pole separation and spindle length establishment. (a) Immunofluorescence of mTPX2^WT, mTPX2^AAA, and mTPX2^ΔN mitotic cells after hTPX2(RNAi), stained for CREST, Cep135, DNA, and α-tubulin. Centrosomes are collapsed to chromatin when the TPX2–Aurora A interaction is abolished. Bar, 10 μm. (b) Centrosome-kinetochore distances are shorter in mTPX2^AAA and mTPX2^ΔN mutants. The centrosome-kinetochore distance from cells stained as in a was determined by measuring individual centrosome-kinetochore distances from metaphase cells in three dimensions (see Materials and methods). For each condition, measurements were taken from four independent cells (eight centrosomes). Red dots show the data points measured and the bars show the means. (c) In TPX2 mutants unable to bind or activate Aurora A, spindle poles collapse immediately after NEBD and only slightly elongate to form shorter metaphase spindles. Distances between spindle poles were measured in three dimensions from live-cell image stacks taken at 1-min intervals of mTPX2^WT, mTPX2^AAA, and mTPX2^ΔN (mTPX2-GFP) cells after hTPX2(RNAi). Thick lines represent means and thin lines represent individual videos. (d) Representative still images from time-lapse recordings quantified in b.