The Kinetochore Protein Kis1/Eic1/Mis19 Ensures the Integrity of Mitotic Spindles through Maintenance of Kinetochore Factors Mis6/CENP-I and CENP-A
Figure 5
Kis1/Mis19 localizes to the kinetochore-SPB interface in interphase.
(A, B) Kis1-GFP was visualized with Sid4-CFP (marks SPB; A) and Mis6-2mRFP (marks kinetochore; B). I: interphase cells; M: mitotic cells. Cell shapes are outlined. (C) Kymograph of Kis1-GFP and Cnp3-tdTomato in a cell filmed from G2 phase to anaphase. Kis1/Mis19 dispersed at the onset of SPB separation (filled arrowhead) and reappeared after chromosome segregation (open arrowhead). The length of the downward arrow corresponds to 2 min. (D) The amount of Kis1-GFP during the cell cycle. The cdc25-22 mutant was used to synchronize the cell cycle at G2. Cells were shifted to 36°C for 4 h and then back to 25°C. Samples were then taken every 20 min (0–120 min). A sample for asynchronous cells (AS) was taken before the temperature shift. Top: Immunoblotting was performed with anti-GFP and anti-α-tubulin (control). Bottom: Kis1-GFP level was normalized with that of α-tubulin (red). Populations of cells with Kis1-GFP dots (green), binucleate cells (blue), and septated cells (black) are also shown. (E) Kis1-GFP was visualized with Sid4-CFP and Mis6-2mRFP in the cdc2-as (analog-sensitive) mutant. The ATP analog 1NM-PP1 was added to cells to inhibit the Cdc2 kinase activity, and images were acquired after 5 min after drug addition (analog, +). A mock-treated cell (analog, −) and cdc2+ cells are also shown. Cells were cultured at 25°C except in (D). Scale bars, 5 µm.
