Supporting Information

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Elaine Franklin
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1 Supporting Information ou et al..73/pnas dd Thymidine Release & transfection dd Thymidine Release dd MG132 Fix and IF -14 h 0 h 8 h 24 h 34 h 36 h siontrol simps1-1 simps1-1 simps1-1 simps1-2 simps1-2 simps1-2 anti-mps1 (~97 K) anti-tubulin (~0 K) [sirn] nm Normalized Mps1 signal simps1-2 siontrol urora N Merge MT N Merge E F simps1 siubr1 siontrol HeLa cells, MT N Merge G siontrol siubr1 simps1 simps1 siubr1 siontrol U2OS cells, siontrol siubr1 simps1 Fig. S1. Mps1 plays a minor role in facilitating chromosome alignment during mitosis. () Schematic representation of the protocol used to analyze the chromosome alignment functions of differently treated cells. () Western blot for Mps1 and α-tubulin of differently treated cells. ells were transfected with siontrol, simps1-1, or simps1-2 at the indicated concentration. Thirty-six hours post transfection, mitotic cell lysates were probed with anti-mps1 and antitubulin antibodies, respectively. () Representative immunofluorescence images of HeLa cells treated with different sirns as indicated. Thirty-six hours post transfection, cells were fixed and costained for aurora (green), (red), and N (blue). (Scale bar, μm.) ( and F) Representative immunofluorescence images of HeLa cells () or U2OS cells (F) treated with different sirns as indicated. Thirty-five hours post transfection, cells were treated with MG132 for 1 h. Then cells were fixed and costained for MT (green), (red), and N (blue). (Scale bars, μm.) (E and G) ar graphs illustrating the percentage of unaligned kinetochores in cells treated as in (E)orF(G). In each experiment, five cells were measured (>60 kinetochores per cell). Values are mean ± SE of three independent experiments. ou et al. 1of7

dd Thymidine Release and cotransfection dd Thymidine Release dd Monastrol Release into MEM+MG132 Fix and IF -14 h 0 h 8 h 24 h 34 h 36 h 37 h MT N Merge shubr1 shmock shmock shubr1 RPE1 cells

2 dd Thymidine Release and cotransfection dd Thymidine Release dd Monastrol Release into MEM+MG132 Fix and IF -14 h 0 h 8 h 24 h 34 h 36 h 37 h MT N Merge shubr1 shmock shmock shubr1 RPE1 cells Monastrol washout into MG132/1h, RPE1 cells Fig. S2. Mps1 is dispensable for the correction of errors in kinetochore MT attachment. () Schematic representation of the protocol used to analyze the functions of kinetochore MT attachment error correction in differently treated cells. () Representative immunofluorescence images of RPE1 cells treated with different shrns as indicated. Thirty-four hours post transfection, cells were treated with monastrol for 2 h and then were released into medium containing MG132 for 1 h. Then cells were fixed and costained for MT (green), (red), and N (blue). (Scale bar, μm.) () ar graph illustrating the percentage of unaligned kinetochores in cells treated as in. Values are mean ± SE of three independent experiments. ou et al. 2of7

MT N Merge MG132/1 h ZM447439 Reversine MSO MSO Rev ZM RPE1 cells MT N Merge MG132/1 h ZM447439 Reversine MSO MSO Rev ZM U2OS cells Fig. S3.

3 MT N Merge MG132/1 h ZM Reversine MSO MSO Rev ZM RPE1 cells MT N Merge MG132/1 h ZM Reversine MSO MSO Rev ZM U2OS cells Fig. S3. Inhibiting Mps1 kinase activity causes a severe defect in chromosome alignment. ( and ) Representative immunofluorescence images of RPE1 cells () and U2OS cells () treated as indicated. ells were fixed and costained for MT (green), (red), and N (blue). (Scale bars, μm.) ( and ) Quantification of the degree of chromosome misalignment in cells treated as in () or (). Kinetochore position was measured as a function of the distance from pole to spindle equator. ars represent the mean ± SE of three independent experiments. In each experiment, five cells were measured (>60 kinetochores per cell). ou et al. 3of7

Reversine MSO LP-Mps1 N Merge kinetochores intensity (a.u.) 1 0 80 60 40 Mps1 Noc+MG132 LP-Mps1 N Merge+ Merge+MT MSO Reversine Mps1-K-8 Mps1-K-8 P=0.006 P=0.

4 Reversine MSO LP-Mps1 N Merge kinetochores intensity (a.u.) Mps1 Noc+MG132 LP-Mps1 N Merge+ Merge+MT MSO Reversine Mps1-K-8 Mps1-K-8 P=0.006 P= Mps1-WT Mps1-K-8 Mps1-K-8 E LP-Mps1 N Merge Reversine MSO Monastrol+MG132 Fig. S4. The kinetochore localization of inactive Mps1 perturbs stable kinetochore MT attachment. () Representative immunofluorescence images of HeLa cells cotransfected with and shrn-resistant constructs as indicated. Thirty-six hours post transfection, cells were treated with MG132 for 1 h and then were immunostained for MT (shown as red in merged images), (shown as red in merged images), and N (blue). (Scale bar, μm.) () Quantification of the degree of chromosome misalignment in cells treated as in. Kinetochore position was measured as a function of the distance from pole to spindle equator. ars represent the mean ± SE of three independent experiments. In each experiment, five cells were measured (>60 kinetochores per cell). () Weakening the kinetochore localization of Mps1 K can rescue the chromosome misalignment phenotype induced by the expression of Mps1 K. Shown are representative immunofluorescence images of cells cotransfected with and shrn-resistant Mps1 constructs as indicated. Thirty-six hours post transfection, cells were treated with MG132 for 1 h and then were immunostained for MT (red), (shown as black and white images), and N (blue). (Scale bar, μm.) () ar graph illustrating the percentage of unaligned kinetochores in cells treated as in. Values are mean ± SE of three independent experiments. (E) Representative immunofluorescence images of LP-Mps1 HeLa cells treated with the inhibitors as indicated and then immunostained for (red) and N (blue). (Scale bar, μm.) ou et al. 4of7

+Monastrol+MG132 E K- 1-14 WT- 1-14 GFP- Mps1-1-61 WT- 1-14 K- 1-14 GFP+ +N GFP- Mps1-1-61 GFP+ +N shmock 62 191 791 1 TPR 0 Kinase 87 664 GFP-Mps1 N Merge +Monastrol+MG132 Mps1-62-2 Mps1-1-2

5 +Monastrol+MG132 E K WT GFP- Mps WT K GFP+ +N GFP- Mps GFP+ +N shmock TPR 0 Kinase GFP-Mps1 N Merge +Monastrol+MG132 Mps Mps1-1-2 Mps Mps1-1-0 Mps Mps1-1-3 GFP-Mps1 N Merge Kinetochore signal intensity (% relative to Mps1-1-3) Mps1-1-3 Mps Mps1-1-0 Mps Mps1-1-2 Mps Fig. S. Inactive Mps1 has a kinetochore localization mechanism distinct from that of Mps1 WT.() Representative immunofluorescence images of HeLa cells cotransfected with GFP-Mps together with or shmock. Thirty-six hours post transfection cells were fixed and costained for (red) and N (blue). (Scale bar, μm.) ( and ) Representative immunofluorescence images of HeLa cells cotransfected with Mps1 shrn and different -resistant plasmids as indicated. Thirty-six hours post transfection cells were treated with indicated drugs for 1 h. Then cells were fixed and costained for (red) and N (blue). (Scale bars, μm.) () ar graph illustrating kinetochore intensity of different Mps1 deletions treated as in. ars represent the mean ± SE of five cells (> kinetochores per cell). (E) Multiple sequence alignment of Mps1 proteins from different species as indicated identifies the conserved RVPV motif within the Mps1 1 0 fragment. The sequence alignment was done by lustalw2 software. ou et al. of7

6 LP-Mps1 N Merge LP-Mps1 N Merge MT ZM Reversine ZM Reversine MSO Nocodazole 2h MSO 1h ctive Mps1 Nocodazole 2h ZM h Monastrol 2h MSO 1h Monastrol 2h ZM h Fig. S6. The kinetochore localization of inactive Mps1 does not stringently require aurora kinase activity. () Representative immunofluorescence images of LP Mps1 HeLa cells treated as indicated. The cells were fixed and costained for (red) and N (blue). (Scale bar, μm.) () The kinetochore localization Legend continued on following page ou et al. 6of7

7 of WT Mps1 depends on aurora activity in both the no MT-attachment situation and the with MT-attachment situation. ells were treated with nocodazole or monastrol for 2 h. Then one group of nocodazole/monastrol-treated cells was treated with ZM for 1 h, and the other group of cells was treated with MSO for 1 h. ells were fixed and costained for (red) and N (blue). (Scale bar, μm.) ou et al. 7of7

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment

Dynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment ynamic localization of Mps1 kinase to kinetochores is essential for accurate spindle microtubule attachment Zhen ou a,b,1, Xing Liu a,b,c,1, Wenwen Wang a,b,c, Tongge Zhu a,b,c, Xinghui Wang a,b, Leilei