Ectopic Activation of the Spindle Assembly Checkpoint Signaling Cascade Reveals Its Biochemical Design

Abstract

Summary Switch-like activation of the Spindle Assembly Checkpoint (SAC) is critical for accurate chromosomes segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the “eSAC”. The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins, and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior.

The Bub1-TPR Domain Interacts Directly with Mad3 to Generate Robust Spindle Checkpoint Arrest

“…Here we bypassed the need for kinetochores and KNL1 Spc105/Spc7 and showed that phosphorylation of Bub1 by Mps1 is sufficient to initiate checkpoint signaling in both budding and fission yeast. While this manuscript was being revised, a study of synthetic checkpoint signaling in HeLa cells described similar findings [44]. As in all spindle checkpoint arrests, we find that a Bub1-Mad1 complex is formed and necessary for effective downstream signaling [9, 45].…”

The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness the Spindle Assembly Checkpoint

“…1A). Even just one MELT motif can delay anaphase when phosphorylated in yeast and human cells (Aravamudhan et al, 2016; Chen et al, 2019; Krenn et al, 2014). Therefore, the number of MELT motifs per Spc105 molecule is a crucial determinant of the strength of SAC signaling, and many MELT motifs per Spc105 are expected to endow it, and by extension the kinetochore, with a larger signaling capacity.…”

“…Indeed, Spc105 and its homologs typically contain many MELT motifs, e.g. six in budding yeast and nineteen in humans (Tromer et al, 2015), and the large MELT motif number is thought to be essential for implementing a strong SAC (Chen et al, 2019; Krenn et al, 2014; Vleugel et al, 2015; Vleugel et al, 2013; Zhang et al, 2014). However, when budding yeast and human cells are treated with high doses of the microtubule poison nocodazole, only a small fraction of MELT motifs, ∼ 20 and ∼ 35% respectively, engage in SAC signaling, and even fewer MELT motifs are sufficient for arresting cell division (Aravamudhan et al, 2016; Vleugel et al, 2015; Vleugel et al, 2013; Zhang et al, 2014).…”

Kinetochore Recruitment of the Spindle and Kinetochore-Associated (Ska) Complex Is Regulated by Centrosomal PP2A in Caenorhabditis elegans

“…Excess Ska could have a negative effect on the SAC, which would decrease survival of rsa-1(or598) by allowing the accumulation of chromosome errors through development. In rheostat-based models of SAC regulation, the signaling strength of the kinetochore adapts to the total number of signaling kinetochores (Collin et al 2013;Chen et al 2019). The chromosome bridges in rsa-1(or598) embryos suggest widespread kinetochore-microtubule attachment errors and, perhaps, this results in a decrease in SAC sensitivity.…”