Journal: Scientific Reports

Article Title: Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation

doi: 10.1038/srep12204

Figure Lengend Snippet: ( a ) Nocodazole-arrested HeLa cells were treated with BI2536 or DMSO for another 1 hr, followed by Western blot analysis with antibodies against pSer715-MCAK, MCAK, pThr210-PLK1, PLK1 and α-tubulin, respectively. ( b ) Images of mitotic HeLa cells treated with BI2536 or DMSO. Cell were fixed and stained with anti-pSer715-MCAK antibody (green), anti-pThr210-PLK1 antibody (red), ACA (blue) and DAPI. Insets on the right show single focal planes of the boxed regions. Scale bars, 10 μm and 1 μm (enlarged images). ( c , d ) HeLa cells were synchronized to G 1 /S boundary by a double thymidine block or prometaphase by Nocodazole treatment. Cells were then harvested and analyzed by Western blotting with the indicated antibodies. Tubulin and MCAK served as a loading control respectively in ( c ) and ( d ). ( e ) Double thymidine treated HeLa cells were released into fresh medium at indicated time. The harvested cells were then analyzed by Western blotting with antibodies against pSer715-MCAK, MCAK, PLK1, CyclinB and α-tubulin (loading control). ( f ) Images of HeLa cells stained with anti-pSer715-MCAK antibody (green), ACA (red), DAPI (blue) and anti-α-tubulin antibody. Representative kinetechores are enlarged and shown on the right . Scale bars, 10 μm and 1 μm (enlarged images).

Article Snippet: Other antibodies were obtained from commercial sources: mouse anti-PLK1 monoclonal antibody (Invitrogen); anti-phospho-Thr210 PLK1 antibody (BD Biosciences); anti-Cyclin B antibody (BD Biosciences); mouse anti-EB1 antibody (BD Biosciences); mouse monoclonal anti-GFP antibody (BD Biosciences); anti-α-tubulin antibody DM1A (Sigma-Aldrich).

Techniques: Western Blot, Staining, Blocking Assay

Journal: Scientific Reports

Article Title: Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation

doi: 10.1038/srep12204

Figure Lengend Snippet: ( a ) HeLa cells synchronized to mitosis by Nocodazole were treated respectively with BI2536, VX-680, ZM447439 or DMSO for another 1 hr, followed by Western blot analysis with the indicated antibodies ( b , e ) Synchronized mitotic HeLa cells were treated with BI2536, ZM447439 or DMSO for 1 hr. Cells were then fixed and stained with anti-pSer715-MCAK antibody or anti-pThr210-PLK1 antibody (green), ACA (red) and DAPI (blue). The enlargements show the representative centromeres. ( c , f ). Statistical analysis of pSer715-MCAK and pThr210-PLK1 immunofluorescence intensity at centromeres in ( b ) and ( e ), respectively. The intensity ratio in DMSO-treated group was normalized to 1. Data are shown as means ± SE and derived from at least 10 cells for each condition. ** P < 0.01, *** P < 0.001, Student’s t -test. ( d ) Recombinant GST-PLK1 was phosphorylated by Aurora kinases. In vitro phosphorylation reactions were performed as described in Methods. GST-PLK1 was visualized by Ponceau S staining and phosphorylation was detected by the antibody against pThr210-PLK1 by Western blotting. ( g ) Color-coded images of HeLa cells expressing a centromere-targeted PLK1 FRET sensor in prometaphase of cells arrested by Taxol treatment. PLK1 inhibitor (GW843682x) or Aurora B inhibitor (Hesperadin) was added at the indicated time point during live-cell imaging. Timestamps relative to drug addition is annotated in min. ( h ) Statistical analysis of the FRET/CFP emission ratio on centromeres at the indicated time ( g ). Data are presented as means ± SE derived from over 100 kinetochores of each category from five different cells. Scale bars, 10 μm (all image panels).

Article Snippet: Other antibodies were obtained from commercial sources: mouse anti-PLK1 monoclonal antibody (Invitrogen); anti-phospho-Thr210 PLK1 antibody (BD Biosciences); anti-Cyclin B antibody (BD Biosciences); mouse anti-EB1 antibody (BD Biosciences); mouse monoclonal anti-GFP antibody (BD Biosciences); anti-α-tubulin antibody DM1A (Sigma-Aldrich).

Techniques: Western Blot, Staining, Immunofluorescence, Derivative Assay, Recombinant, In Vitro, Expressing, Live Cell Imaging

Journal: Scientific Reports

Article Title: Spatiotemporal dynamics of Aurora B-PLK1-MCAK signaling axis orchestrates kinetochore bi-orientation and faithful chromosome segregation

doi: 10.1038/srep12204

Figure Lengend Snippet: ( a ) Color-coded images of HeLa cells expressing PLK1 or Aurora B sensor show chromosome alignment after Syntelin washout. Cells were treated with 1 μM Syntelin for 30 min followed by three washes for subsequent real-time imaging analyses. Timestamps are in minutes. ( b ) Quantitative analysis of FRET/CFP emission ratio on centromeres relative to the metaphase plate. Schematic illustration of centromere position calculation (see also ). ( c ) HeLa cells were treated with MG132 for 1 hr to allow for metaphase plate formation. Parallel samples were then treated with 1 μM Taxol or DMSO for 40 min before staining for PLK1 (green), Aurora B (red) and ACA (blue). Insets show individual kinetochore pairs used for line scans. ( d ) Live-cell imaging of chromosome segregation in GFP-MCAK constructs-addback cells. Cells were treated according to the protocol outlined in the upper panel. Arrows indicate lagging chromosomes during segregation. Timestamps in hr:min. ( e ) Quantitative analysis of the defective anaphases seen in ( d ). Data are presented as means ± SD from three independent experiments. * P < 0.05, Student’s t -test. ( f ) Cold-stable KT-MT attachment in mCherry-MCAK mutants-addback cells. Cells treated with Monastrol were released into MG132-containing medium for another 1 hr, and then fixed and stained for MTs (green), ACA (red) and DAPI (blue). Arrows indicate erroneous kinetochore attachments. ( g ) Quantitative analysis of cells exhibiting one or more aberrant attachments in various mutant MCAK-expressing cells. Data are presented as means ± SD from three independent experiments. * P < 0.05, Student’s t -test. ( h ) Model of accurate regulation of MCAK by Aurora B-PLK1 axis at kinetochore. Aurora B locates closely to PLK1 at prophase, enabling PLK1 to be activated by Aurora B at inner-centromeres (upper row). During the prometaphase-to-metaphase transition, stretch on kinetochore pairs may separate PLK1 and MCAK from Aurora B to outer-kinetochores. The stimulated MCAK activity ensures error correction in KT-MT attachment (middle row). Once the amphitelic attachments are generated, both activities of PLK1 and MCAK are down-regulated, and anaphase begins with chromosomes segregating towards the opposite poles (lower row). Scale bars, 10 μm (all image panels).

Article Snippet: Other antibodies were obtained from commercial sources: mouse anti-PLK1 monoclonal antibody (Invitrogen); anti-phospho-Thr210 PLK1 antibody (BD Biosciences); anti-Cyclin B antibody (BD Biosciences); mouse anti-EB1 antibody (BD Biosciences); mouse monoclonal anti-GFP antibody (BD Biosciences); anti-α-tubulin antibody DM1A (Sigma-Aldrich).

Techniques: Expressing, Imaging, Staining, Live Cell Imaging, Construct, Mutagenesis, Activity Assay, Generated