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Fig. 2 | BMC Molecular and Cell Biology

Fig. 2

From: Aurora A site specific TACC3 phosphorylation regulates astral microtubule assembly by stabilizing γ-tubulin ring complex

Fig. 2

S558A mutation in TACC3 affects correct positioning of mitotic centrosomes. a. Time-lapse images of live metaphase-synchronized HeLa Kyoto cells stably expressed with α-tubulin-GFP and H2B-mCherry were transfected with i) GFP-TACC3 (WT)-TACC3 shRNA, ii) GFP-TACC3 (S558A)-TACC3 shRNA or iii) GFP-TACC3 (S558D)-TACC3 shRNA are shown. The time at which chromosomes were visibly aligned to the metaphase plate (based on the H2B mCherry staining) after thymidine release refers to t = 0 min. GFP and bright-field images of the same cell are shown in top and bottom panel, respectively in each. Respective time-lapse movies are shown in Additional file 1: Movie S1, Additional file 2: Movie S2 and Additional file 3: Movie S3. b. Times of metaphase to anaphase progression in TACC3 WT, S558A and S558D expressed mitotic cells are plotted. Data are mean +/− S. E. (three experiments) c. Since the cells imaged in A are also expressed with α-tubulin-GFP, the expressions of the GFP-tagged TACC3 variants were confirmed by Western blot. GFP-TACC3 constructs (WT or the mutants) were expressed in all the three conditions. d. The bright-field time lapse images of HeLa Kyoto cells transfected with i) GFP-TACC3 (WT)-TACC3 shRNA, or ii) GFP-TACC3 (S558A)-TACC3 shRNA followed by thymidine treatment and release thereafter, were imaged soon after the cells entered mitosis after thymidine release (t = 00:00 h:min). The images of the same mitotic cells from the start to end of mitosis are shown. It was confirmed by snapshot GFP imaging (at t = 0) that the cells that were imaged were expressed with the respective GFP-TACC3 proteins (shown in first image in each captured at t = 0, GFP is displayed in grey colour). The respective time-lapse movies are shown in Additional file 4: Movie S4, Additional file 5: Movie S5. e. The plot shows total mitotic progression time in WT vs. S558A mutant condition. Data are mean +/− S. E. (three experiments)

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