Kops GJ , Kim Y , Weaver BA , Mao Y , McLeod I , Yates JR , Tagaya M , Cleveland DW .

GM 29513 NIGMS NIH HHS , R37 GM029513-22 NIGMS NIH HHS , RR11823-09 NCRR NIH HHS , R37 GM029513 NIGMS NIH HHS , R01 GM029513 NIGMS NIH HHS , P41 RR011823 NCRR NIH HHS

	Figure 1. ZW10 and Zwint-1 reside in distinct kinetochore subcomplexes in HeLa cells. (A) Localization of ZW10LAPtag in a HeLa cell line stably expressing the fusion protein (clone LZ5). Cells were treated with nocodazole for 30 min before fixation, and stained for ZW10LAPtag (anti-GFP), centromeres (ACA), and DNA (DAPI). (B) Localization of Zwint-1LAPtag in clone LINT2.8. Treatment and staining was performed as in A, except cells were preextracted before fixation. (C) Immunoblot of whole cell lysates of HeLa cells and clone LZ5. Lysates were probed for ZW10, ZW10LAPtag (anti-GFP), and tubulin. (D and E) Tandem affinity purification of Zwint-1LAPtag (D) and ZW10LAPtag (E) from mitotically arrested cells. 25% of eluate was analyzed by SDS-PAGE followed by silverstain and 75% was analyzed by MudPIT mass spectrometry. Name, percent sequence coverage, and expected molecular weight of the identified proteins are indicated in the table. Suspected position of the identified proteins on the silver stained gel are indicated on the right. Unlabeled bands on silverstain likely include DC31 (∼32 kD), Q9H410 (∼40 kD), and the nonspecific proteins HSP70 (∼70 kD) and α-tubulin (∼50 kD).
	Figure 2. Interaction between Zwint-1 and ZW10 controls ZW10 kinetochore localization. (A) Immunoblot of Zwint-1 immunoprecipitates shows weak interaction with ZW10. Cells of clone LINT2.8 were subjected to immunoprecipitation with control antibody (Con) or anti-GFP antibody to precipitate Zwint-1LAPtag and the precipitate was analyzed for the presence of endogenous ZW10. HSS, high speed supernatant before the immunoprecipitation. Bands labeled with asterisks are background due to precipitation from HSS with the anti-GFP antibody. White line indicates that intervening lanes have been spliced out. (B) Analysis of Zwint-1 knockdown efficiency by immunoblot using cells expressing Zwint-1LAPtag. Lysates of LINT2.8 cells untransfected or transfected with mock or Zwint-1 siRNA plasmid for 72 h were analyzed for Zwint-1LAPtag (anti-GFP), ZW10, and tubulin expression. Percentage of remaining protein was determined by serial dilution immunoblotting. Band labeled with asterisks is protein that cross reacts with anti-GFP in the LINT2.8 cell line. (C) Immunolocalization of ZW10 in cells depleted of endogenous Zwint-1. HeLa cells transfected as in B were treated with nocodazole for 30 min before fixation and stained for endogenous Zwint-1 and ZW10, and for centromeres (ACA) and DNA (DAPI).
	Figure 3. Characterization of Xenopus ZW10 and Rod. (A and B) Schematic alignment of Xenopus and human ZW10 (A) or Xenopus RodCOOH (XL107l09) and human Rod (B). Amino acid positions as well as percentage identity and additional (*) similarity on the protein level are indicated. (C) Coomassie staining of purified recombinant X-ZW10 (X-Z) and X-RodCOOH (X-RCOOH). (His)6-tagged proteins were purified from insect cells and analyzed by Coomassie blue staining. (D) Immunoblot analysis of pAbs to X-ZW10 and X-RodCOOH. 20 ng of recombinant protein (rec. prot.) and 1 μl CSF extract were analyzed by immunoblot with affinity purified anti–X-ZW10 (1348) or anti–X-RodCOOH (1351). Position of the endogenous frog proteins in the CSF extract is indicated. Cross-reacting proteins are marked by asterisks. (E) Immunolocalization of X-ZW10 and X-Rod. Sperm nuclei replicated in cycled CSF extract were immunostained for X-BubR1 and X-ZW10 or X-Rod. DNA (DAPI) is in blue. Enlarged boxes show overlap of X-BubR1 and X-ZW10–X-Rod signals on a sister kinetochore pair.
	Figure 4. The X-ZW10–X-Rod complex is essential for establishment and maintenance of the mitotic checkpoint. (A) Immunoblot of immunodepleted CSF extract. Extracts were depleted with anti–rabbit IgG (ΔIgG), anti–X-ZW10 (ΔX-Z), or anti–X-RodCOOH (ΔX-R). 1 μl of extract or the eluate of 1-μl beads from the immunodepletion were analyzed for X-ZW10–X-Rod levels. (B) Cdk1 kinase activity in Xenopus oocyte extracts. CSF extracts, mock depleted (ΔIgG) or depleted of the X-ZW10–X-Rod complex (ΔX-ZW10), were supplemented with nocodazole and the indicated amount of sperm nuclei and mitotic checkpoint activity was measured by the ability to maintain Cdk1 kinase activity toward histone H1 (H1) after inactivation of CSF by calcium for 0, 30, or 60 min. White lines indicate that intervening lanes have been spliced out. (C) CSF extracts were supplemented with sperm (15,000 per μl of extract) before (maintenance) or after (establishment) mock depletion (ΔIgG) or depletion of the X-ZW10–X-Rod complex (ΔX-ZW10 or ΔX-Rod). Mitotic checkpoint activity was measured as in B.
	Figure 5. X-ZW10–X-Rod regulate kinetochore localization of X-BubR1, X-Mad1, and X-Mad2. (A–C) Immunolocalization of checkpoint proteins in depleted Xenopus extracts. Unreplicated sperm nuclei in mock (A, ΔIgG) or X-ZW10–X-Rod–depleted (B, ΔX-ZW10; or C, ΔX-Rod) extracts were immunostained with antibodies to the indicated proteins and X-BubR1. DNA (DAPI) is in blue. (D) Immunoblot of various checkpoint proteins in Xenopus extracts depleted of the X-ZW10–X-Rod complex. CSF extracts or checkpoint activated extracts were mock depleted (ΔIgG) or depleted of the X-ZW10–X-Rod complex (ΔX-Z or ΔX-R) and analyzed for presence of the indicated checkpoint proteins. (E) Immunolocalization of X-ZW10 and X-Rod in X-BubR1–depleted extracts. CSF extracts, mock depleted (ΔIgG) or depleted of X-BubR1 (ΔX-BubR1) were analyzed for kinetochore localization of X-ZW10 and X-Rod. DNA (DAPI) is in blue.
	Figure 6. Human ZW10 is essential for mitotic checkpoint signaling. (A) Immunoblot analysis of ZW10 knockdown. HeLa cells were transfected with control or ZW10 siRNA duplexes. 5 d after transfection, total lysates were analyzed for ZW10 and tubulin protein. Percentage knockdown was determined by serial dilution immunoblotting. (B) Immunofluorescence analysis of ZW10 knockdown. Cells were transfected as in A, treated with nocodazole for 30 min before fixation and stained for ZW10 and DNA (DAPI). (C) Flow cytometric analysis of the fraction of phospho-histone H3-positive cells of mock siRNA, ZW10 siRNA and Zwint-1 siRNA cells 96 h after introduction of the siRNAs. Cells were transfected as in A, left untreated or treated with nocodazole for 16 h, and the entire population was analyzed for phospho-histone-H3 (y axis) and DNA (propidium iodide, x axis). Dot plots represent 4×104 cells for control siRNA and 104 cells for ZW10 or Zwint-1 siRNA. Percentages indicate fraction of cell population that is phospho-histone H3 positive. (D) Average fold increase of phospho-histone H3 staining after nocodazole treatment. Cells were transfected, treated, and analyzed as in C. Graph represents average of four independent experiments. (E) Graph of colony outgrowth assay. Cells were transfected as in A and retransfected for 7 d after which the colonies were stained and counted. Graph represents average of three experiments. (F and G) Aberrant mitosis in cells lacking ZW10. Cells were transfected as in A and stained with DAPI. Shown are typical interphase nuclei. (F) Cells transfected with ZW10 siRNA. (G) Cells transfected with control siRNA.
	Figure 7. Mad1 and Mad2 are unable to bind kinetochores in absence of ZW10. (A–F) Immunolocalization of various proteins in ZW10-depleted cells. HeLa cells were transfected as in Fig. 6 A and treated with nocodazole 30 min before fixation. Cells were stained with ACA (A, B, and C), ZW10 (A, B, D, and F), and dynein intermediate chain (IC) (A), Mad1 (B), Mad2 (C), Bub1 (D), or BubR1 (F). DNA (DAPI) is in blue. Enlarged boxes show a pair of kinetochores. (E) Quantification of kinetochore fluorescence. Normalized integrated intensity (see Materials and methods) of Mad1, Mad2, and Bub1 is shown in mock siRNA and ZW10 siRNA cells. Error bars indicate the SD from measurements of three cells. (G) Model for function of the ZW10–Rod complex in mitotic checkpoint signaling. The ZW10-interactor Zwint-1 is in a structural kinetochore complex with Ndc80–HEC1 and Mis12 that is linked to the inner kinetochore by KNL-1AF15q14. The ZW10–Rod–Zwilch complex associates dynamically with the unattached kinetochore through interaction with Zwint-1, where it regulates kinetochore binding of the Mad1–Mad2 heterodimer and thus activation of Mad2.

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