Mps1 Regulates Kinetochore-Microtubule Attachment Stability via the Ska Complex to Ensure Error-Free Chromosome Segregation

Abstract

SUMMARY The spindle assembly checkpoint (SAC) kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1’s error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K-fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule-sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K-fibers in vivo and reduced the efficiency of the Ska complex’s conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation.

Microtubule Tip Tracking by the Spindle and Kinetochore Protein Ska1 Requires Diverse Tubulin-Interacting Surfaces

“…At low Ska1 complex concentration, we observed that individual molecules associated with and tracked the microtubule plus end only briefly, indicating that this tracking is not highly processive (Figure 3B). Additionally, although work from others has not detected plus end tracking of elongating microtubules by the Ska1 complex [13], we observed a notably increased brightness for GFP-Ska1 complex fluorescence at growing microtubule ends (Figure 3B; Figure S3C; Movie S1) indicative of plus end tracking with microtubule polymerization.…”

“…In addition, although the Ska1 MTBD is necessary and sufficient for Ska1 complex microtubule binding and spindle localization [2], we note that recent work has also suggested a contribution to microtubule interactions from Ska3 [28]. Ultimately, these activities must be regulated [13, 29, 30] and integrated with other players at the kinetochore, including the Ndc80 complex [2, 31, 32], to ensure faithful chromosome segregation during every cell division.…”

The RZZ complex integrates spindle checkpoint maintenance with dynamic expansion of unattached kinetochores

“…S1A-D). Cell extracts were prepared by nocodazole treatment, mitotic shakeoff, and nitrogen cavitation as described [3,23,34] and immunoprecipitated with Zw10-specific antibodies. Samples were resolved by All was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.…”

“…3C). We identified two Mps1 phosphorylation sites at the N-terminus of Rod (T13 and S15), upstream of its beta-propeller domain [23]. To test their function, wildtype (WT) and nonphosphorylatable (2A) Rod were expressed in T-Rex FLP-in HeLa cells as LAP (EGFP-TEV-S-peptide) fusions (Fig.…”

“…In light of these findings and the fact that Mps1 maximally phosphorylates Rod at unattached kinetochores [23], we propose that this modification overcomes the barrier to Spindly-RZZ polymerization and kinetochore expansion. Thereafter end-on microtubule attachments reverse this phosphorylation, in part through counteracting PP2A-B56 phosphatases [23], thus reinhibiting Spindly-RZZ and facilitating kinetochore compaction. In summary, we have identified a key trigger for dynamic kinetochore expansion that involves SACAll rights reserved.…”