Abstract
The spindle assembly checkpoint is a surveillance mechanism that blocks anaphase onset until all chromosomes are properly attached to microtubules of the mitotic spindle. Checkpoint activity requires kinetochore localization of Mad1/Mad2 to inhibit activation of the anaphase promoting complex/cyclosome in the presence of unattached kinetochores. In budding yeast and Caenorhabditis elegans, Bub1, recruited to kinetochores through KNL1, recruits Mad1/Mad2 by direct linkage with Mad1. However, in human cells it is not yet established which kinetochore protein(s) function as the Mad1/Mad2 receptor. Both Bub1 and the RZZ complex have been implicated in Mad1/Mad2 kinetochore recruitment; however, their specific roles remain unclear. Here, we investigate the contributions of Bub1, RZZ and KNL1 to Mad1/Mad2 kinetochore recruitment. We find that the RZZ complex localizes to the N-terminus of KNL1, downstream of Bub1, to mediate robust Mad1/Mad2 kinetochore localization. Our data also point to the existence of a KNL1-, Bub1-independent mechanism for RZZ and Mad1/Mad2 kinetochore recruitment. Based on our results, we propose that in humans, the primary mediator for Mad1/Mad2 kinetochore localization is the RZZ complex.
Highlights
The spindle assembly checkpoint (SAC) senses attachment between kinetochores and the mitotic spindle, and prevents anaphase until all kinetochores are properly connected to spindle microtubules (MTs)
We do not believe that the Mad1 remaining at kinetochores after Bub1 RNAi is due to incomplete Bub1 depletion since Bub1 was not detected at kinetochores by immunofluorescence in these cells co-stained for Mad1 and Bub1, and treatment of Bub1-depleted cells with nocodazole does not result in accumulation of detectable kinetochore-associated Bub1 [15]
We observed kinetochore Mad1 in KNL1-depleted cells in the presence of nocodazole. These results demonstrate that Mad1 can be recruited at low levels to kinetochores in the absence of Bub1 and, surprisingly, in the absence of KNL1. We propose that both Bub1 and the RZZ complex are required for robust Mad1 kinetochore recruitment, but, while recruitment via Bub1 is dispensable for low levels of Mad1 localization, recruitment via RZZ is necessary for the localization of Mad1 to kinetochores, even at low levels
Summary
The spindle assembly checkpoint (SAC) senses attachment between kinetochores and the mitotic spindle, and prevents anaphase until all kinetochores are properly connected to spindle microtubules (MTs). The protein Zwint, which binds the C-terminal region of KNL1, is proposed to recruit the RZZ complex, which is implicated in Mad kinetochore recruitment [9,10,11,12]. Together, this evidence suggests that KNL1 mediates Mad recruitment in a bimodal manner, through its N-terminal region via Bub1/Bub and through its C-terminal region via RZZ/Zwint1 [4,5,6,13,14,15,16]. The mechanism by which Bub and the RZZ complex contribute to Mad kinetochore localization, and the potential link between these two recruitment pathways, are still unclear.
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