Fig 2: Structural analysis & size-exclusion chromatography of the Aurora-A/TACC3 complex Size-exclusion chromatographs of Aurora-A and TACC3 alone and in complex on a Superdex 200 16/600 column (GE Healthcare).Electron density map of the Aurora-A/TACC3 complex. Aurora-A is coloured cyan. TACC3 is coloured orange. The 2mFo-DFc electron density map is shown as a wire mesh contoured at 1.0 s.Side chain HC chemical shift perturbations observed for 13C15N TACC3 519–540 on the addition of Aurora-AKD D274N.Small section showing mainly methyl group resonances from a 13C-HSQC spectrum of TACC3 519–540. The spectrum for TACC3 519–540 is shown in blue. The spectrum collected on the addition of Aurora-AKD D274N is coloured red.

Fig 3: Defects in spindle localization and late mitotic progression in CRISPR/Cas9-engineered HeLa cells with mutations in TACC3 APLA showing Aurora-A/TACC3 (top panel) and Aurora-A/TPX2 (bottom panel) complexes during mitosis in HeLa cells. Cell cycle stages are indicated above cell images.BDot plot indicates volume of TACC3 protein at spindle pole regions of WT and F525A HeLa cells in telophase. Volumes were measured for 90 spindle pole regions per cell line (45 telophase cells). Representative images are shown in Fig EV4C.C, DPLA between Aurora-A and TACC3 in WT, ?TACC and F525A HeLa cells. Representative metaphase (C), and anaphase and telophase cells (D) are shown in panels on the left. Box and whisker plots on the right represent the number of PLA dots counted per cell in either metaphase or anaphase/telophase cells, respectively. 30 cells were analysed for each cell cycle stage for each cell line.EDuration of anaphase to chromosome decondensation, measured by time-lapse microscopy in WT, ?TACC and F525A HeLa cells. Number of cells analysed is indicated on graph (n).Data information: Cell images include DNA stained with DAPI (blue). Scale bars, 10 µm. In the dot plots (B, E), horizontal lines represent the mean and error bars correspond to standard deviation. P-values were obtained from Mann–Whitney: ****P < 0.0001. For the box and whisker plots (C, D), the middle horizontal line marks the median and the box represents the 25th and 75th percentile. The whiskers extend from the minimum to the maximum values. P-values were obtained from Student's t-test, unpaired, two-tailed: ****P < 0.0001.

Fig 4: In vitro kinase assay – CPEB1 phosphorylation.(A) Oocytes were cultivated in control medium for 0, 28 or 44 h, Thereafter, in vitro kinase assay with CPEB1 peptide as an external substrate was performed in extracts prepared from 20 oocytes. MLN8237 was added to MI (28 h of IVM) oocyte extracts in final concentration of 10 µM in reaction buffer. Alternatively, recombinant AURKA (1 and 5 I.U. per assay) was used instead of oocyte extracts with or without MLN8237 in the concentration of 2 and 10 µM. The extracts were separated by SDS-PAGE and the phosphorylated substrates were visualized by autoradiography and analyzed using a BAS 2500 PhosphoImager. (B) In vitro kinase assay utilizing recombinant AURKA (1 I.U.) with or without MLN8237 in the concentration of 2 and 10 µM showing its activity towards either CPEB1 peptide as an external substrate, or LATS2 peptide used as a positive control. Phosphorylated substrates were separated by SDS-PAGE, visualized by autoradiography and analyzed using a BAS 2500 PhosphoImager.

Fig 5: Phosphorylation by Aurora A regulates the phase separation status and function of NuMA in mitosis.a Immunofluorescence images showing the localization and features of GFP-NuMA-dNLS droplets in interphase HeLa cells transfected with GFP-NuMA-dNLS with or without nocodazole treatment. b, c Time-lapse images and quantification of FRAP of GFP-NuMA-dNLS droplets in mitotic HeLa cells without (b) or with nocodazole (c) treatment (mean ± SEM, n = 10 droplets per sample). Scale bars, 2 µm. d Phase separation of GFP-CT in interphase cytoplasmic extracts and mitotic cytoplasmic extracts under the indicated conditions, followed by visualization with confocal microscopy. e Quantification of FRAP of phase-separated GFP-CT droplets in interphase cytoplasmic extracts and mitotic extracts (mean ± SEM, n = 10 droplets per sample). f Immunofluorescence images of NuMA droplets in HeLa cells, showing the different morphologies. The cells were treated without (Ctrl) or with MLN8237, followed by treatment with nocodazole and staining with anti-NuMA antibody (gray). g Quantification of FRAP of endogenous NuMA-mClover droplets in the control or MLN8237-treated NuMA-mACF cells (mean ± SEM, n = 5 droplets for control and n = 11 droplets for MLN8237-treated cells). h Confocal microscopic images of GFP-CT or GFP-CT-S1969A droplets phase-separated in vitro in the mentioned conditions. GFP-CT or GFP-CT-S1969A proteins were incubated with kinase buffer containing Aurora A/ATP or Aurora A alone, followed by visualization with confocal microscopy. i Quantification of FRAP of GFP-CT or GFP-CT-S1969A droplets in (h) (mean ± SEM, n = 10, 8, 10, and 8 droplets for WT + ATP, WT-ATP, S1969A + ATP, and S1969A-ATP, respectively). j Time-lapse images of stable RFP-H2B-expressing HeLa cells transfected with GFP, GFP-NuMA, GFP-S1969A, and GFP-S1969D. Images were collected every 4 min. The white arrows indicate the aberrant congression of chromosomes. k The average time from NEBD to anaphase onset of the NuMA-expressing HeLa cells in j (mean ± SEM, n = 14, 15, 15, and 24 cells were measured for GFP, NuMA, S1969A, and S1969D, respectively). Unpaired two-tailed t test: P = 0.1723 for GFP/NuMA, P = 0.0083 for NuMA/S1969A, P = 0.2211 for NuMA/S1969D, P = 0.0006 for S1969A/S1969D. **P < 0.01, and ***P < 0.001, ns, not significant. Scale bars, 10 µm unless specified otherwise.