Journal: The Journal of Cell Biology

Article Title: MAD1-dependent recruitment of CDK1-CCNB1 to kinetochores promotes spindle checkpoint signaling

doi: 10.1083/jcb.201808015

Figure Lengend Snippet: PP2A-B55 opposes CCNB1 recruitment to kinetochores. (A) Checkpoint signaling and CCNB1 localization were followed in control (siControl) and PP2A-B55 (siB55) depleted HeLa CCNB1-GFP cells. Cells were arrested for 2.5 h with 20 µM MG132, and then either fixed immediately (+MG132) or treated with 3 µM nocodazole for 5 min (+Noc) or with 5 µM flavopiridol for 1 min followed by addition of 3 µM nocodazole for 5 min (+CDK-i +Noc). MAD1 and kinetochores (CREST) were detected using antibodies, and CCNB1 using GFP fluorescence. (B) Mean kinetochore intensity ± SEM of CCNB1 and MAD1 in control (siCon) and B55-depleted (siB55) cells are plotted (15 kinetochores per cell for ≥5 cells in each of three independent experiments). (C) A schematic of the checkpoint response assay. (D) Control (siControl) and PP2A-B55 (siB55) depleted HeLa cells arrested in mitosis for 3 h with 100 µM monastrol were washed into fresh growth medium for 25 min to allow spindle formation. At that point, 0 min, or after a further 10 or 20 min, cells were challenged with 3 µM nocodazole for 5 min to test for the checkpoint response, fixed, and then stained for MAD1 and tubulin. (E) Graphs show the fraction of checkpoint silenced cells at different times after monastrol washout, before nocodazole addition. Bars indicate the SEM (for siControl: 0/10/20 min n = 263/293/277 and for siB55: n = 296/294/277). (F) Graphs show the fraction of checkpoint active cells with unseparated sister chromatids (for siControl: 0/10/20min n = 232/302/297 and for siB55: n = 277/257/306) and MAD1 signal at kinetochores (for siControl: 0/10/20min n = 64/48/65 and for siB55: n = 46/62/49); error bars indicate the SEM. (G) CCNB1 and MPS1 localization are shown for the 20-min time point challenged with 3 µm nocodazole (+Noc) in control (siCon) or PP2A-B55 (siB55) depleted HeLa CCNB1-GFP/MPS1-mCherry cells. MPS1 intensity is plotted relative to the cytoplasmic CCNB1 signal for individual kinetochores with the mean and SD (15 kinetochores per cell in 26 [siCon] or 27 [siB55] cells).

Article Snippet: Inhibitor stocks prepared in DMSO were as follows: 5 mM CDK-inhibitor flavopiridol (Sigma-Aldrich), 20 mM MPS1-inhibitor AZ3146 (Tocris Bioscience), 100 mM Eg5 inhibitor Monastrol (Cambridge Bioscience), 20 mM proteasome inhibitor MG132 (Insight Bioscience), and 6 mM microtubule polymerization inhibitor nocodazole (Merck).

Techniques: Fluorescence, Staining

Journal: bioRxiv

Article Title: Checkpoint signaling and error correction require regulation of the MPS1 T-loop by PP2A-B56

doi: 10.1101/626937

Figure Lengend Snippet: (A) MPS1 WT or GFP-MPS1 DD mitotic cells were stained for DNA (grey), MAD1 and CENP-C (red in the merged image). MPS1 was visualised by GFP fluorescence. An enlarged image of kinetochore pairs, indicated by a dashed box, is shown on the right-hand side of the images. (B) The x-y distance of individual kinetochores from the cell centre in 23 (MPS1 WT ) or 19 (MPS1 DD ) randomly selected cells is plotted. (C) Mean ± SEM proportion of misaligned, MAD1 positive or MPS1 positive kinetochores in the cells measured in (B) . (D) GFP-MPS1 WT or MPS1 DD expressing cells were filmed after Monastrol washout; images were captured every 2 min. Scale bar, 10 μm. (E) Quantification of cells from (D) . Each horizontal bar represents a single cell. (F) GFP-MPS1 expressing cells were fixed following a Monastrol washout into MG132. MPS1i was added at 2 μM for 60 min alongside MG132 where indicated. Images show DNA (grey), microtubules (red) and GFP-MPS1 (green). Bar graphs show mean proportion ± SD of cells exhibiting aligned metaphase plates. (G) KNL1-KNL1 inter-kinetochore distance is shown in GFP-MPS1 WT or GFP-MPS1 DD cells treated as in (A) . Each dot represents a kinetochore pair, and the black bar represents the mean average of 234 (WT) and 290 (DD) kinetochore pairs across 19 cells per condition from 2 independent experiments. (H) Cells as in (A) were cold treated prior to fixation. DNA (grey) and microtubules (red). The mean proportions ± SD of cells (282 WT and 331 DD cells from three independent experiments) containing bipolar spindles with aligned metaphase plates or cells with <10 stable microtubule-kinetochore attachments are plotted.

Article Snippet: Inhibitors were obtained from Tocris Bioscience (MPS1-inhibitor AZ3146 20mM stock; PP1 and PP2A-inhibitors calyculin A 1mM stock, PP1-inhibitor tautomycetin, 2.5mM stock), Insight Bioscience (proteasome inhibitor MG132 20mM stock), Merck (microtubule polymerisation inhibitor nocodazole 6mM stock) and Cambridge Bioscience (Eg5 inhibitor Monastrol, 100mM stock).

Techniques: Staining, Fluorescence, Expressing