A: Mitotic progression in a representative wild-type cell expressing Atb2-GFP. B: Mitotic progression in a representative tip1Δ cell. Hoechst staining of the DNA is shown at 10, 12, and 16 minutes. C: Mitotic progression in a representative tea2Δ cell. D: Left panel; Mitotic progression in a representative mal3Δ cell. E: Statistical analysis of the length of time spent in metaphase in wild-type, mal3Δ, tea2Δ, and tip1Δ cells. The time spent in metaphase was calculated for each cell as the time interval required for the spindle to progress from 2 µm in length to the onset of anaphase B. The means are represented by the red bars. F: Statistical analysis of the length of time spent in metaphase in the mad2Δ and bub1Δ strains, as well as the various double deletions of the +TIP genes and either the mad2 or bub1 genes. The red lines represent the mean length of time spent in metaphase in the parental lines (wild-type, mal3Δ, tea2Δ and tip1Δ), while the blue bars represent the mean length of time spent in metaphase in the double delete. The grey circles represent the interval of confidence for each analysis with p<0.05.

A: Sequential images of a wild-type cell, expressing Tip1-GFP and Cdc11-CFP (SPB), during mitosis. B: Sequential images of mitosis in a wild-type cell co-expressing Tubulin-RFP and Tip1-GFP. C: Upper panels; Images of two metaphase wild-type cells co-expressing Tubulin-RFP and Tip1-GFP. Lower panels; Line-scans of the plane through the length of the spindle in early mitosis (left panel) or later in mitosis (right panel). The red lines represent the length of the spindle, while the green traces represent the intensity of the Tip1-GFP signal. D: Graphical representation of the timing of appearance of Tip1-GFP at the SPBs during mitosis in four representative cells (Time 0 is anaphase onset). Each trace plots the length of the spindle versus time. Filled circles; Times at which Tip1-GFP is associated with either of the two SPBs.

A: Upper panel; Representative field of mal3Δ Tip1-GFP cells stained with Hoechst (green, Tip1-GFP; red, Hoechst). Lower panel; Representative field of mal3Δ Tea2-GFP cells stained with Hoechst (green, Tea2-GFP; red, Hoechst). B: Upper panel; Representative field of tip1Δ Mal3-GFP cells stained with Hoechst (green, Mal3-GFP; red, Hoechst). Lower panel; Representative field of tip1Δ Tea2-GFP cells stained with Hoechst (green, Tea2-GFP; red, Hoechst). C: Upper panel; Representative field of tea2Δ Tip1-GFP cells stained with Hoechst (green, Tip1-GFP; red, Hoechst). Lower panel; Representative field of tea2Δ Mal3-GFP cells stained with Hoechst (green, Mal3-GFP; red, Hoechst).

A: Sensitivity of the mal3Δ, tea2Δ and tip1Δ strains to TBZ. B: Rate of chromosome loss in the +TIPΔ strains as compared to wild-type and checkpoint gene delete strains.

A: Each strain carried epitope tagged versions of the core kinetochore component Ndc80 (green, K) and the SPB protein Cdc11 (Cdc11-CFP in red, S). B: Upper panel; Sequential images of a wild-type cell expressing Ndc80-GFP and Cdc11-CFP during mitosis. Middle panel; Kymograph representation of the same cell. Lower panel; Graphical analysis of the movements of the SPBs (red) and the kinetochores (green) in this cell, determined by automated analysis. C: Upper panel; Sequential images of a tip1Δcell expressing Ndc80-GFP and Cdc11-CFP during mitosis. Middle panel; Kymograph representation of the same cell. Lower panel; Graphical analysis of the movements of the SPBs (red) and the kinetochores (green).

A: Upper panel; Sequential images of a mal3Δ cell expressing Ndc80-GFP and Cdc11-CFP during mitosis. Kymograph representation of the same cell. Graphical analysis of the movements of the SPBs (red) and the kinetochores (green). B: Upper panel; Sequential images of a tea2Δ cell expressing Ndc80-GFP and Cdc11-CFP during mitosis. Middle panel; Kymograph representation of the same cell. Lower panel; Graphical analysis of the movements of the SPBs (red) and the kinetochores (green).

A: Percentage of cells entering anaphase A with one or more lagging chromosomes. B: Repartition of lagging chromosomes as a function of their lag times in a logarithmic scale. C: Rate of spindle elongation during anaphase B in the wild type and tip1Δ cells. The empty circles represent mitosis without lagging chromosomes present, while the filled circles represent mitosis with lagging chromosomes. The mean spindle elongation rate (red bars) was calculated for each strain and were not found to be significantly different (student test).

Left-hand panels; Metaphase cells. Right-hand panels; Anaphase cells with lagging chromosomes. A: The upper block shows images of an early metaphase tip1Δ cell, expressing Mad2-Cherry (red) and Ndc80-GFP (green). The lower block shows a cell in late metaphase. The line-scans of the merged images is shown on the right. The position of the line taken for the scans is marked by the blue crosses. B: Kymograph showing the localization of Mad2 during progression through metaphase in a tip1Δ Mad2-Cherry Ndc80-GFP cell. C: Images of an anaphase tip1Δ Mad2-Cherry Ndc80-GFP cell with lagging chromosomes. D: Images of a metaphase tip1Δ cell, expressing Bub1-GFP (green) and Ndc80-CFP (red). E: Images of an anaphase tip1Δ Bub1-GFP Ndc80-CFP cell showing lagging chromosomes. F: Kymograph showing the localization of Bub1 during progression through metaphase in a tip1Δ Bub1-GFP Ndc80-CFP cell. G: Kymograph showing the localization of Bub1 during progression through anaphase in a tip1Δ Bub1-GFP Ndc80-CFP cell.