The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A
Figure 7
Kis1/Mis19 regulates Mis6 for spindle integrity.
(A) Cells coexpressing Mis6-2GFP and Cnp3-tdTomato were imaged. (B,C) ChIP assays. Percentages of DNA precipitates of the centromeric region (cnt and imr) and the arm region (lys1) were measured as scaled to total input DNA using quantitative PCR. Black bars (+), immunoprecipitation with anti-GFP; gray bars (−), mock-treated negative control. Error bars indicate standard deviation (n = 3 reactions) (B) ChIP of Mis6-2GFP in WT or in kis1-1 cells grown at 36°C for 6 h. (C) ChIP of Kis1-GFP. (D) Localization of Kis1-GFP in WT and mis6-302 cells grown at 36°C for 6 h. Percentages indicate the proportion of interphase cells in which Kis1-GFP colocalized with both Cnp3-tdTomato and Sid4-CFP. (E) Ten-fold serial dilution assay. WT or kis1-1 cells expressing either GFP (−) or Mis6-GFP (mis6) were grown on EMM plates at 25, 30, or 32°C. (F) A proposed model for localization dependency of Kis1/Mis19, Mis6 and Cnp1/CENP-A at kinetochores. (G) (a,b) Spindle phenotypes of mis6-302 cells. Time-lapse imaging of GFP-Atb2, Nup40-mCherry, and Sid4-CFP in mis6-302 was done at 36°C (6–9 h). (a) Blob spindles; (b) spindles with the dim midzone; (c) frequencies of spindle defects in WT, kis1-1, and mis6-302 cells. Scale bars, 5 µm.
