FIGURE 8:
Retrieval of unclustered kinetochores delays the onset of anaphase and requires PP1Dis2. (A) Images from two representative movies of mitotic Δnsk1 fta3-gfp sid4-tdtomato cells that entered mitosis with either clustered kinetochores (top panels) or an unclustered kinetochore (middle panels). Time zero is the point of pole separation. Images from a representative movie of a mitotic Δnsk1 Δdis2 fta3-gfp sid4-tdtomato cell that entered mitosis with an unclustered kinetochore pair (bottom panels). This cell was already in mitosis when imaging began. Images were taken at 1-min intervals. Arrows indicate unclustered kinetochores. (B) Movies of Δnsk1 fta3-gfp sid4-tdtomato and Δdis2 Δnsk1 fta3-gfp sid4-tdtomato cells with unclustered kinetochores during mitosis were analyzed, and the distance (“d” in the schematic) from kinetochore (green) to SPB (red) was measured in each frame. Blue lines represent retrieval events in cells that subsequently go through anaphase. Red lines represent the paths of kinetochores in cells where spindles subsequently collapse (see Figure 8A and Movie S5). Green and purple lines represent kinetochores that are retrieved but subsequently lost and retrieved again before proceeding into anaphase. Note: we cannot confirm whether the same kinetochores are lost in each case. (C) The length of time individual fta3-gfp sid4-tdtomato and Δnsk1 fta3-gfp sid4-tdtomato cells spent in prometaphase and metaphase (PM + M) was determined by live-cell imaging. Cells lacking Nsk1 were divided into those that entered mitosis with at least one unclustered kinetochore (unclustered) and those that did not (clustered). Closed circles represent movies where mitotic entry and exit can be assessed. Open circles represent movies where either entry or exit from mitosis are absent. The mean duration of completed mitoses from each class is represented by a horizontal line. (D) Log-phase cultures of wild-type, Δnsk1, Δdis2, and Δdam1 cells expressing nda3-KM311 dad1-gfp sid4-tdtomato were arrested in mitosis, as before. Kinetochore recapture was assayed as described in Materials and Methods. Error bars are the SD from the mean of three independent experiments.