Dual regulation of Mad2 localization on kinetochores by Bub1 and Dam1/DASH that ensure proper spindle interaction

Abstract

The spindle assembly checkpoint monitors the state of spindle-kinetochore interaction to prevent premature onset of anaphase. Although checkpoint proteins, such as Mad2, are localized on kinetochores that do not interact properly with the spindle, it remains unknown how the checkpoint proteins recognize abnormalities in spindle-kinetochore interaction. Here, we report that Mad2 localization on kinetochores in fission yeast is regulated by two partially overlapping but distinct pathways: the Dam1/DASH and the Bub1 pathways. We show that Mad2 is localized on "unattached" as well as "tensionless" kinetochores. Our observations suggest that Bub1 is required for Mad2 to detect tensionless kinetochores, whereas Dam1/DASH is crucial for Mad2 to detect unattached kinetochores. In cells lacking both Bub1 and Dam1/DASH, Mad2 localization on kinetochores is diminished, and mitotic progression appears to be accelerated despite the frequent occurrence of abnormal chromosome segregation. Furthermore, we found that Dam1/DASH is required for promotion of spindle association with unattached kinetochores. In contrast, there is accumulating evidence that Bub1 is involved in resolution of erroneous spindle attachment on tensionless kinetochores. These pathways may act as molecular sensors determining the state of spindle association on each kinetochore, enabling proper regulation of the checkpoint activation as well as promotion/resolution of spindle attachment.

Figure 1.

Mono-oriented capture is not sufficient for release of Mad2 from the kinetochore. (A) Snapshot image of an nda3 mutant cell expressing Mad2-YFP, Mis12-Cherry (a kinetochore component), and CFP-Atb2 (α-tubulin) after recovery from mitotic arrest. Nine serial Z-sections with an interval of 0.3 μm were flattened after deconvolution. The position of the kinetochore detached from the spindle is indicated by an arrowhead. In the merged panel, Mad2-YFP, Mis12-Cherry, and CFP-Atb2 are pseudocolored green, red, and blue, respectively. Bar, 10 μm. (B) Time-lapse images of an nda3 cell expressing Mad2-YFP and CFP-Atb2 after recovery from mitotic arrest. Panels show the YFP channel alone (Mad2), and YFP merged with CFP (merge: YFP and CFP are pseudocolored green and magenta, respectively). Eleven serial Z-sections with an interval of 0.4 μm were collected every 20 s, deconvolved, and flattened using a Leica ASMDW microscopy system. The bright Mad2 spot coincident with the detached/mono-oriented kinetochore is indicated by an arrowhead. The numbers indicate the duration in seconds. Bar, 10 μm. (C) The fluorescence intensity of the Mad2-YFP spot was measured at each time point using three-dimensional images of the cell shown in B and is plotted in arbitrary units (left axis). Vertical distance between the Mad2 spot and the spindle was also calculated (right axis).

Figure 2.

Bub1 is required for retention of Mad2 on mono-oriented kinetochores. Time-lapse images of cut9 Δbub1 cells expressing Mad2-GFP and CFP-Atb2 after removal of CBZ. Panels show the GFP channel (Mad2) and GFP merged with CFP (merge: GFP and CFP are colored green and magenta, respectively). Stacks of Z-sections with an interval of 0.4 μm were taken every 20 s. Three-dimensional images were flattened after deconvolution. Two cells are shown in each panel and the outlines of the cells are drawn in the panel of time = 0; the upper cell is in interphase, and the other is in mitosis. The Mad2 spot presumably coincident with a detached kinetochore is indicated by an arrowhead. The numbers indicate the duration in seconds. Bar, 10 μm.

Figure 3.

Bub1 is indispensable for localization of Mad2 on cohesion-defective kinetochores. The cut9 single mutant, cut9 mis4 double mutant, and cut9 mis4 Δbub1 triple mutant cells were incubated at the restrictive temperature for two generations (5, 6, and 8 h, respectively), and fixed for immunostaining of Cnp1 (s.p. CENP-A). Mad2 was visualized by GFP fusion in these cells. Stacks of Z-sections were taken and flattened after deconvolution. In merge panels, Mad2, Cnp1, and DNA are colored green, red, and blue, respectively. Bar, 5 μm.

Figure 4.

Δbub1 Δhos2 double gene deletion impairs accumulation of Mad2 on unattached kinetochores. (A) Cells with the indicated genotype were treated with CBZ after S phase synchronization. Details are described in the text. Images of Mad2-GFP in these cells are shown, and bright Mad2 spots are indicated by arrowheads. Bar, 10 μm. Calculated percentages of cells with the Mad2 spot in the mitotic population are presented in the right panel. (B) Percentages of cells with Mad2 spot in total cells were counted every 15 min after addition of CBZ. More than 250 cells were examined for each sample. Cells were presynchronized in S phase as described in the text. (C) Snapshot of an nda3 mutant cell expressing Hos2-YFP, Mis12-Cherry (a kinetochore component) and CFP-Atb2 (α-tubulin) after recovery from mitotic arrest. Nine serial Z-sections with an interval of 0.3 μm were flattened after deconvolution. The position of the kinetochore detached from the spindle is indicated by an arrowhead. In the merged panel, Hos2-YFP, Mis12-Cherry, and CFP-Atb2 are pseudocolored green, red, and blue, respectively. Bar, 10 μm. (D) Spindle lengths from multiple movies of exponentially growing cells with the indicated genotype were analyzed. Cells were cultured in minimal medium at 26°C, and Pcp1-CFP (SPB marker) was used to determine the length. The time point at which phase 3 spindle extension began was defined as 0. The number of samples examined (n) and average time required for phase 1 + 2 (average ± SD) in each strain are shown.

Figure 5.

Δhos2 cells are defective in poleward movement of kinetochores during anaphase A. Time-lapse images of mitosis in exponentially growing cells with the indicated genotype are presented. Kinetochores and the SPB were visualized by Mis12-GFP (green) and Pcp1-CFP (magenta), respectively. Cells were cultured in minimal medium at 26°C, and stacks of Z-sections with an interval of 0.4 μm were taken every 0.5 min. The numbers indicate the duration in minutes. The time point at which phase 3 spindle extension began was defined as 0. Bar, 10 μm.

Figure 6.

Model: Bub1 and Dam1/DASH may coordinate the checkpoint control with error-correcting machineries. Mad2 accumulation on kinetochores are regulated by two distinct pathways: the Dam1/DASH pathway and the Bub1 pathway. Unattached kinetochores are thought to be detected by the Dam1/DASH pathway, which may trigger Mad2 accumulation on the kinetochores and also facilitate spindle–kinetochore attachment. On the other hand, tensionless kinetochores are detected by the Bub1 pathway, which may promote both Mad2 accumulation on the kinetochore and the resolution of tensionless spindle attachment. This resolution of the attachment results in conversion of the tensionless kinetochore into the unattached kinetochore.