Abstract
SummaryThe spindle checkpoint acts as a mitotic surveillance system, monitoring interactions between kinetochores and spindle microtubules and ensuring high-fidelity chromosome segregation [1, 2, 3]. The checkpoint is activated by unattached kinetochores, and Mps1 kinase phosphorylates KNL1 on conserved MELT motifs to generate a binding site for the Bub3-Bub1 complex [4, 5, 6, 7]. This leads to dynamic kinetochore recruitment of Mad proteins [8, 9], a conformational change in Mad2 [10, 11, 12], and formation of the mitotic checkpoint complex (MCC: Cdc20-Mad3-Mad2 [13, 14, 15]). MCC formation inhibits the anaphase-promoting complex/cyclosome (Cdc20-APC/C), thereby preventing the proteolytic destruction of securin and cyclin and delaying anaphase onset. What happens at kinetochores after Mps1-dependent Bub3-Bub1 recruitment remains mechanistically unclear, and it is not known whether kinetochore proteins other than KNL1 have significant roles to play in checkpoint signaling and MCC generation. Here, we take a reductionist approach, avoiding the complexities of kinetochores, and demonstrate that co-recruitment of KNL1Spc7 and Mps1Mph1 is sufficient to generate a robust checkpoint signal and prolonged mitotic arrest. We demonstrate that a Mad1-Bub1 complex is formed during synthetic checkpoint signaling. Analysis of bub3Δ mutants demonstrates that Bub3 acts to suppress premature checkpoint signaling. This synthetic system will enable detailed, mechanistic dissection of MCC generation and checkpoint silencing. After analyzing several mutants that affect localization of checkpoint complexes, we conclude that spindle checkpoint arrest can be independent of their kinetochore, spindle pole, and nuclear envelope localization.
Highlights
Co-tethering KNL1Spc7 and Mps1Mph1 Kinase Generates a Robust Mitotic Arrest At unattached kinetochores, Bub1 is thought to recruit Mad1 [20]
When we expressed the phosphomimic mutant Spc7(1-666)-9TE fused to the Tet repressor in these cells it resulted in constitutive recruitment of Bub1, Bub3, and Mad3 to the tet operator (tetO) array, throughout the cell cycle and independently of Mph1 kinase (Figure 1B)
Bub1p, Bub3p, and Mad3p are recruited to the array with the expected dependencies: we believe that this Spc7-Bub-Mad3 complex likely acts as an independent signaling module (Figure 1C)
Summary
Sadhbh Soper Nı Chafraidh, Eliska Zlamalova , Kevin G. Yuan et al employ synthetic protein assemblies to generate a robust spindle checkpoint arrest in fission yeast. Formation of artificial heterodimers between a checkpoint kinase and one of its key substrates is shown to be sufficient for mitotic arrest, entirely independently of the location of this complex in the yeast nucleus. Highlights d Synthetic signaling scaffolds generate a spindle checkpoint arrest d The combination of KNL1Spc and Mps1Mph kinase generates a robust arrest d Kinetochore, spindle, and nuclear envelope enrichment of the scaffold is not required d Bub acts to inhibit premature checkpoint activation. 2017, Current Biology 27, 137–143 January 9, 2017 a 2017 The Author(s).
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