While SAC signaling is essential to ensure sufficient time for all chromosomes to attach to the spindle, equally important is that signaling be extinguished at each kinetochore upon microtubule binding. Indeed, kinetochore-based SAC signaling is intimately influenced by the binding of microtubules at the kinetochore. For example, several SAC proteins that are enriched at unattached kinetochores are depleted upon microtubule attachment, including Mad1, and Mad2 (reviewed in 55
). Depletion of SAC proteins from kinetochores attached to spindle microtubules is likely to be a mechanistically relevant step in SAC extinction because constitutive targeting of Mad1 to the kinetochore is sufficient to sustain Mad2-dependent SAC signaling after chromosome bi-orientation57
. Although the molecular mechanisms that couple microtubule binding to silencing of the SAC remain largely unclear, here we describe recent studies indicating that microtubule binding status is relayed through the KMN network to couple microtubule attachment to changes in SAC chemistry.
Considering that SAC activation at the kinetochore is dependent on phosphorylation, it is reasonable to suppose that a phosphatase might play a role in SAC extinction. Indeed, SAC extinction at the kinetochore has recently been shown to depend on recruitment of protein phosphatase 1 (PP1)135, 136
(). PP1 is targeted to the kinetochore via a PP1-binding motif present in Knl1 (), indicating that the KMN network, and Knl1 in particular, contributes both to SAC activation and extinction. Deletion of Knl1’s PP1 interaction motif is lethal in budding yeast, due to failure to silence the SAC137
. Viability can be rescued by fusing wild type, but not a catalytically dead mutant of PP1 directly to Knl1, arguing that dephosphorylation of kinetochore substrates contributes to SAC silencing. Similarly, in fission yeast and C. elegans
, disruption of Knl1-dependent kinetochore targeting of PP1 compromises SAC inactivation138, 139
Molecular mechanisms of SAC extinction at the kinetochore
In C. elegans
, a second Knl1-dependent mechanism of SAC silencing has recently been identified. At the extreme N-terminus of Knl1 resides a microtubule-binding domain2
(). Unexpectedly, the short stretch of basic amino acids, adjacent to the PP1-binding motif, is not required for chromosome-spindle attachments in vivo
, but rather contributes to SAC silencing138
. Moreover, loss of both the microtubule-binding activity and PP1 targeting motif in Knl1 yields additive defects on SAC silencing, suggesting the two routes to SAC extinction are independent.
In human cells, the Knl1-PP1 interaction has thus far only been linked to stabilization of kinetochore-microtubule attachment107
. A role for PP1 in SAC extinction may be obscured by redundant, Knl1-independent, mechanisms to silence kinetochore-based SAC signaling, in particular dynein-dependent removal of SAC regulators from the kinetochore140, 141
. Dynein ‘strips’ proteins away from kinetochores towards microtubule minus ends, including Mad1, Mad2, and Spindly, a protein that promotes microtubule binding at the kinetochore140, 141
. Of these, dynein-dependent removal of Spindly is important for SAC extinction at the kinetochore because removal of other cargo, such as Mad1 and Mad2, can also be achieved through dynein-independent, perhaps KMN network-dependent, mechanisms whereas Spindly removal can not140
. It is not clear why the continued presence of Spindly at kinetochores interferes with SAC extinction. Spindly is not required for SAC signaling, and so its removal is unlikely to be directly related to SAC extinction. Rather, the removal of Spindly may be important for other activities at the kinetochore, such as the transition from lateral to tip-based microtubule attachments.
These recent insights are consistent with the notion that Knl1 is both a ‘scaffold’ for SAC activation and extinction (through recruitment of Bub1 and PP1, respectively) and a ‘sensor’, coupling microtubule binding to SAC extinction. How either extinction pathway is relayed to downstream SAC components remains unclear. Identifying substrates of PP1 at the kinetochore will be necessary to understand its role in SAC silencing, with phosphorylation sites in Knl1 as obvious candidates.
Finally, extinction of SAC signaling at the kinetochore must also be integrated with liberation of Cdc20 from its inhibitors Mad2, Bub3, and Mad3/BubR1 to activate the APC/C. APC/C-dependent ubiquitylation of Cdc20 plays a conserved role in disassembling MCC complexes50, 142–144
, and, in metazoans, an additional protein, p31 (comet), a binding partner of ‘closed’ Mad2145, 146
, promotes both disassembly of the MCC and ubiquitylation of Cdc20144, 147, 148
. It will be important to examine whether microtubule capture at the kinetochore accelerates MCC disassembly, perhaps through phospho-Knl1, which, in addition to SAC activation, has also been linked to timely SAC silencing82
. Understanding how the balance of SAC kinase and phosphatase activity at the kinetochore is altered upon microtubule binding to allow cell cycle progression is clearly an important area for future work.