Editing Spindle checkpoint (section)

== Spindle assembly checkpoint overview ==
The spindle assembly checkpoint (SAC) is an active signal produced by improperly attached [[kinetochores]], which is conserved in all [[eukaryotes]]. The SAC stops the cell cycle by negatively regulating CDC20, thereby preventing the activation of the polyubiquitynation activities of [[anaphase promoting complex]] (APC). The proteins responsible for the SAC signal compose the [[mitotic checkpoint complex]] (MCC), which includes SAC proteins, [[MAD2]]/[[MAD3]] (mitotic arrest deficient), [[BUB3]] (budding uninhibited by benzimidazole), and [[CDC20]].<ref name = "Mad2">{{cite journal | vauthors = De Antoni A, Pearson CG, Cimini D, Canman JC, Sala V, Nezi L, Mapelli M, Sironi L, Faretta M, Salmon ED, Musacchio A | title = The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint | journal = Current Biology | volume = 15 | issue = 3 | pages = 214–25 | date = February 2005 | pmid = 15694304 | doi = 10.1016/j.cub.2005.01.038 | s2cid = 3224122 | doi-access = free | bibcode = 2005CBio...15..214D }}</ref> Other proteins involved in the SAC include [[MAD1]], [[BUB1]], [[MPS1]], and [[Aurora B kinase|Aurora B]]. For higher eukaryotes, additional regulators of the SAC include constituents of the [[ZW10|ROD-ZW10 complex]], [[p31comet|p31<sup>comet</sup>]], [[Kinesin|MAPK]], [[CDK1|CDK1-cyclin-B]], [[NEK2]], and [[PLK1]].<ref name="Nature"/>

[[File:Spindle checkpoint vertebrates - en.png|600 px|Scheme representing the different components of the spindle checkpoint in vertebrates.]]

=== Checkpoint activation ===

The SAC monitors the interaction between improperly connected kinetochores and spindle [[microtubules]], and is maintained until kinetochores are properly attached to the spindle. During [[prometaphase]], CDC20 and the SAC proteins concentrate at the kinetochores before attachment to the spindle assembly. These proteins keep the SAC activated until they are removed and the correct kinetochore-microtubule attachment is made. Even a single unattached kinetochore can maintain the spindle checkpoint.<ref name="Mad2"/> After attachment of microtubule plus-ends and formation of kinetochore microtubules, MAD1 and MAD2 are depleted from the kinetochore assembly. Another regulator of checkpoint activation is kinetochore tension. When sister kinetochores are properly attached to opposite spindle poles, forces in the mitotic spindle generate tension at the kinetochores. Bi-oriented sister kinetochores stabilize the kinetochore-microtubule assembly whereas weak tension has a destabilizing effect. In response to incorrect kinetochore attachments such as [[Kinetochore#Verification of kinetochore-MT anchoring|syntelic]] attachment, where both kinetochores becomes attached to one spindle pole, the weak tension generated destabilizes the incorrect attachment and allows the kinetochore to reattach correctly to the spindle body. During this process, kinetochores that are attached to the mitotic spindle but that are not under tension trigger the spindle checkpoint. Aurora-B/Ipl1 kinase of the [[chromosomal passenger complex]] functions as the tensions sensor in improper kinetochore attachments. It detects and destabilizes incorrect attachments through control of the microtubule-severing KINI kinesin MCAK, the [[DASH complex]], and the [[NDC80|Ndc80/Hec1]] complex<ref name="MartinLluesma2002">{{cite journal | vauthors = Martin-Lluesma S, Stucke VM, Nigg EA | title = Role of Hec1 in spindle checkpoint signaling and kinetochore recruitment of Mad1/Mad2 | journal = Science | volume = 297 | issue = 5590 | pages = 2267–70 | date = September 2002 | pmid = 12351790 | doi = 10.1126/science.1075596 | bibcode = 2002Sci...297.2267M | s2cid = 7879023 }}</ref>  at the microtubule-kinetochore interface.<ref name="Nature"/> The Aurora-B/Ipl1 kinase is also critical in correcting [[Kinetochore#Verification of kinetochore-MT anchoring|merotelic]] attachments, where one kinetochore is simultaneously attached to both spindle poles. Merotelic attachments generate sufficient tension and are not detected by the SAC, and without correction, may result in chromosome mis-segregation due to slow chromatid migration speed. While microtubule attachment is independently required for SAC activation, it is unclear whether tension is an independent regulator of SAC, although it is clear that differing regulatory behaviors arise with tension.

Once activated, the spindle checkpoint blocks [[anaphase]] entry by inhibiting the [[anaphase-promoting complex]] via regulation of the activity of mitotic checkpoint complex. The mechanism of inhibition of APC by the mitotic checkpoint complex is poorly understood, although it is hypothesized that the MCC binds to APC as a [[pseudosubstrate]] using the [[KEN-box]] motif in [[BUBR1]]. At the same time that mitotic checkpoint complex is being activated, the [[centromere]] protein [[CENPE|CENP-E]] activates BUBR1, which also blocks anaphase.<ref name="Nature"/>

=== Mitotic checkpoint complex formation ===

The mitotic checkpoint complex is composed of [[BUB3]] together with MAD2 and MAD3 bound to [[Cdc20]]. MAD2 and MAD3 have distinct binding sites on CDC20, and act synergistically to inhibit APC/C. The MAD3 complex is composed of BUB3, which binds to Mad3 and [[BUB1B]] through the [[short linear motif]] known as the GLEBS motif. The exact order of attachments which must take place in order to form the MCC remains unknown. It is possible that Mad2-Cdc20 form a complex at the same time as BUBR1-BUB3-Cdc20 form another complex, and these two subcomplexes are consequently combined to form the mitotic checkpoint complex.<ref name="Mad2"/> In human cells, binding of BUBR1 to CDC20 requires prior binding of MAD2 to CDC20, so it is possible that the MAD2-CDC20 subcomplex acts as an initiator for MCC formation. BUBR1 depletion leads only to a mild reduction in Mad2-Cdc20 levels while Mad2 is required for the binding of BubR1-Bub3 to Cdc20. Nevertheless, BUBR1 is still required for checkpoint activation.<ref name="Nature"/>

The mechanism of formation for the MCC is unclear and there are competing theories for both kinetochore-dependent and kinetochore-independent formation. In support of the kinetochore-independent theory, MCC is detectable in ''[[S. cerevisiae]]'' cells in which core kinetocore assembly proteins have been mutated and cells in which the SAC has been deactivated, which suggests that the MCC could be assembled during mitosis without kinetochore localization. In one model, unattached prometaphase kinetochores can 'sensitize' APC to inhibition of MCC by recruiting the APC to kinetochores via a functioning SAC. Furthermore, depletions of various SAC proteins have revealed that MAD2 and BUBR1 depletions affect the timing of mitosis independently of kinetochores, while depletions of other SAC proteins result in a dysfunctional SAC without altering the duration of mitosis. Thus it is possible that the SAC functions through a two-stage timer where MAD2 and BUBR1 control the duration of mitosis in the first stage, which may be extended in the second stage if there are unattached kinetochores as well as other SAC proteins.<ref name="Nature"/> However, there are lines of evidence which are in disfavor of the kinetochore-independent assembly. MCC has yet to be found during [[interphase]], while MCC does not form from its constituents in ''[[X. laevis]]'' [[meiosis II]] extracts without the addition of sperm of nuclei and [[nocodazole]] to prevent spindle assembly.

The leading model of MCC formation is the "MAD2-template model", which depends on the kinetochore dynamics of MAD2 to create the MCC. MAD1 localizes to unattached kinetochores while binding strongly to MAD2. The localization of MAD2 and BubR1 to the kinetochore may also be dependent on the [[Aurora B kinase]].<ref>{{cite journal | vauthors = Lens SM, Wolthuis RM, Klompmaker R, Kauw J, Agami R, Brummelkamp T, Kops G, Medema RH | title = Survivin is required for a sustained spindle checkpoint arrest in response to lack of tension | journal = The EMBO Journal | volume = 22 | issue = 12 | pages = 2934–47 | date = June 2003 | pmid = 12805209 | pmc = 162159 | doi = 10.1093/emboj/cdg307 }}</ref> Cells lacking Aurora B fail to arrest in metaphase even when chromosomes lack microtubule attachment.<ref>{{cite journal | vauthors = Hauf S, Cole RW, LaTerra S, Zimmer C, Schnapp G, Walter R, Heckel A, van Meel J, Rieder CL, Peters JM | title = The small molecule Hesperadin reveals a role for Aurora B in correcting kinetochore-microtubule attachment and in maintaining the spindle assembly checkpoint | journal = The Journal of Cell Biology | volume = 161 | issue = 2 | pages = 281–94 | date = April 2003 | pmid = 12707311 | pmc = 2172906 | doi = 10.1083/jcb.200208092 }}</ref> Unattached kinetochores first bind to a MAD1-C-MAD2-p31<sup>comet</sup> complex and releases the p31<sup>comet</sup> through unknown mechanisms. The resulting MAD1-C-MAD2 complex recruits the open conformer of Mad2 (O-Mad2) to the kinetochores.  This O-Mad2 changes its conformation to closed Mad2 (C-Mad2) and binds Mad1.  This Mad1/C-Mad2 complex is responsible for the recruitment of more O-Mad2 to the kinetochores, which changes its conformation to C-Mad2 and binds Cdc20 in an auto-amplification reaction. Since MAD1 and CDC20 both contain a similar MAD2-binding motif, the empty O-MAD2 conformation changes to C-MAD2 while binding to CDC20. This [[positive feedback loop]] is negatively regulated by p31<sup>comet</sup>, which competitively binds to C-MAD2 bound to either MAD1 or CDC20 and reduces further O-MAD2 binding to C-MAD2. Further control mechanisms may also exist, considering that p31<sup>comet</sup> is not present in lower eukaryotes.  The 'template model' nomenclature is thus derived from the process where MAD1-C-MAD2 acts as a template for the formation of C-MAD2-CDC20 copies. This sequestration of Cdc20 is essential for maintaining the spindle checkpoint.<ref name="Mad2"/>

=== Checkpoint deactivation ===

Several mechanisms exist to deactivate the SAC after correct bi-orientation of [[sister chromatids]]. Upon microtubule-kinetochore attachment, a mechanism of stripping via a [[dynein|dynein-dynein motor complex]] transports spindle checkpoint proteins away from the kinetochores.<ref name = "Nature">{{cite journal | vauthors = Musacchio A, Salmon ED | title = The spindle-assembly checkpoint in space and time | journal = Nature Reviews. Molecular Cell Biology | volume = 8 | issue = 5 | pages = 379–93 | date = May 2007 | pmid = 17426725 | doi = 10.1038/nrm2163 | s2cid = 205494124 }}</ref> The stripped proteins, which include MAD1, MAD2, MPS1, and [[CENPF|CENP-F]], are then redistributed to the [[spindle pole]]s. The stripping process is highly dependent on undamaged microtubule structure as well as dynein motility along microtubules. As well as functioning as a regulator of the C-MAD2 positive feedback loop, p31<sup>comet</sup> also may act as a deactivator of the SAC. Unattached kinetochores temporarily inactivate p31<sup>comet</sup>, but attachment reactivates the protein and inhibits MAD2 activation, possibly by inhibitory phosphorylation. Another possible mechanism of SAC inactivation results from energy-dependent dissociation of the MAD2-CDC20 complex through non-degradative ubiquitylation of CDC20. Conversely, the de-ubiquitylating enzyme [[protectin]] is required to maintain the SAC. Thus, unattached kinetochores maintain the checkpoint by continuously recreating the MAD2-CDC20 subcomplex from its components. The SAC may also be deactivated by APC activation induced [[proteolysis]]. Since the SAC is not reactivated by the loss of sister-chromatid cohesion during anaphase, the proteolysis of cyclin B and inactivation of the CDK1-cyclin-B kinase also inhibits SAC activity. Degradation of MPS1 during anaphase prevents the reactivation of SAC after removal of sister-chromatid cohesion. After checkpoint deactivation and during the normal anaphase of the cell cycle, the anaphase promoting complex is activated through decreasing MCC activity. When this happens the enzyme complex [[Polyubiquitination|polyubiquitinates]] the anaphase inhibitor [[securin]]. The ubiquitination and destruction of securin at the end of metaphase releases the active protease called separase. Separase cleaves the cohesion molecules that hold the sister chromatids together to activate anaphase.<ref name = "Morgan">{{Cite book | edition = 1 | publisher = New Science Press, Ltd | isbn = 978-0-87893-508-6 | last = Morgan | first = David O. | title = The Cell Cycle: Principles of Control (Primers in Biology) | date = 2006-09-06 }}</ref>

==== New model for SAC deactivation in ''S. cerevisiae'': the mechanical switch ====
A new mechanism has been suggested to explain how end-on microtubule attachment at the kinetochore is able to disrupt specific steps in SAC signaling.  In an unattached kinetochore, the first step in the formation of the MCC is phosphorylation of Spc105 by the kinase Mps1.  Phosphorylated Spc105 is then able to recruit the downstream signaling proteins Bub1 and 3; Mad 1,2, and 3; and Cdc20.  Association with Mad1 at unattached kinetochores causes Mad2 to undergo a conformational change that converts it from an open form (O-Mad2) to a closed form (C-Mad2.)  The C-Mad2 bound to Mad1 then dimerizes with a second O-Mad2 and catalyzes its closure around Cdc20.  This C-Mad2 and Cdc20 complex, the MCC, leaves Mad1 and C-Mad2 at the kinetochore to form another MCC.  The MCCs each sequester two Cdc20 molecules to prevent their interaction with the APC/C, thereby maintaining the SAC.<ref name="Morgan" />  Mps1's phosphorylation of Spc105 is both necessary and sufficient to initiate the SAC signaling pathway, but this step can only occur in the absence of microtubule attachment to the kinetochore.  Endogenous Mps1 is shown to associate with the calponin-homology (CH) domain of Ndc80, which is located in the outer kinetochore region that is distant from the chromosome.  Though Mps1 is docked in the outer kinetochore, it is still able to localize within the inner kinetochore and phosphorylate Spc105 because of flexible hinge regions on Ndc80.  However, the mechanical switch model proposes that end-on attachment of a microtubule to the kinetochore deactivates the SAC through two mechanisms. The presence of an attached microtubule increases the distance between the Ndc80 CH domain and  Spc105. Additionally, Dam1/DASH, a large complex consisting of 160 proteins that forms a ring around the attached microtubule, acts as a barrier between the two proteins.  Separation prevents interactions between Mps1 and Spc105 and thus inhibits the SAC signaling pathway.<ref>{{cite journal | vauthors = Aravamudhan P, Goldfarb AA, Joglekar AP | title = The kinetochore encodes a mechanical switch to disrupt spindle assembly checkpoint signalling | journal = Nature Cell Biology | volume = 17 | issue = 7 | pages = 868–79 | date = July 2015 | pmid = 26053220 | pmc = 4630029 | doi = 10.1038/ncb3179 }}</ref>

This model is not applicable to SAC regulation in higher order organisms, including animals.  A main facet of the mechanical switch mechanism is that in ''S. cerevisiae'' the structure of the kinetochore only allows for attachment of one microtubule. Kinetochores in animals, on the other hand, are much more complex meshworks that contain binding sites for a multitude of microtubules.<ref>{{Cite book|title=Molecular Biology of The Cell (6th ed.)|vauthors = Alberts B, Johnson A, Lewis J, Morgan D, Raff M, Roberts K,Walter P|publisher=Garland Science, Taylor & Francis Group|year=2015|isbn=978-0-8153-4432-2|location=New York, NY|pages=988}}</ref>  Microtubule attachment at all of the kinetochore binding sites is not necessary for deactivation of the SAC and progression to anaphase.  Therefore, microtubule-attached and microtubule-unattached states coexist in the animal kinetochore while the SAC is inhibited.  This model does not include a barrier that would prevent Mps1 associated with an attached kinetochore from phosphorylating Spc105 in an adjacent unattached kinetochore. Furthermore, the yeast Dam1/DASH complex is not present in animal cells.