Cell Science at a Glance 4139 The spindle checkpoint mechanism that delays anaphase onset highlight current understanding of how until all chromosomes are correctly the spindle checkpoint is activated, how it Karen M. May and Kevin G. attached in a bipolar fashion to the delays anaphase onset, and how it is Hardwick mitotic spindle. silenced. Wellcome Trust Centre for Cell Biology, University of Edinburgh, EH9 3JR, UK The core spindle checkpoint proteins are (e-mail: [email protected]) Mad1, Mad2, BubR1 (Mad3 in yeast), Activation of the checkpoint Journal of Cell Science 119, 4139-4142 Bub1, Bub3 and Mps1. The Mad and Bub During mitosis spindle microtubules Published by The Company of Biologists 2006 proteins were first identified in budding bind to complex protein structures called doi:10.1242/jcs.03165 yeast by genetic screens for mutants that kinetochores, which assemble on the failed to arrest in mitosis when the spindle centromere of each chromosome. The Every mitosis, replicated chromosomes was destroyed (Taylor et al., 2004). These Mad and Bub proteins localise to the must be accurately segregated into each proteins are conserved in all eukaryotes. outer kinetochore early in mitosis, before daughter cell. Pairs of sister chromatids Several other checkpoint components, proper attachments are established, and attach to the bipolar mitotic spindle such as Rod, Zw10 and CENP-E, have accumulate on unattached kinetochores. during prometaphase, they are aligned at since been identified in higher eukaryotes When spindle microtubules make metaphase, then sisters separate and but have no yeast orthologues (Karess, contact with the outer kinetochore are pulled to opposite poles during 2005; Mao et al., 2003). This reflects a a number of complex molecular anaphase. Failure to attach correctly to more complex checkpoint regulation in interactions take place that regulate both the spindle before anaphase onset results higher eukaryotes where, unlike in yeasts, attachment and microtubule dynamics in unequal segregation of chromosomes, checkpoint proteins are essential and (Maiato et al., 2004). The checkpoint which can lead to cell death or disease. regulate normal mitotic timing (Meraldi et proteins are therefore ideally placed to The spindle checkpoint is a surveillance al., 2004; Taylor et al., 2004). Here, we monitor these interactions. The Spindle Checkpoint Karen M. May and Kevin G. Hardwick Core checkpoint proteins Mad2 Mad1 Binds constitutively to Mad2 and recruits it to the kinetochore; also localises Mad2 to the nuclear periphery (NP) in interphase, function at the NP is unknown; binds to Amphitelic Bub1 and Bub3 upon checkpoint activation in budding yeast. O C Mad2 Binds to Mad1 and Cdc20; exists in two conformations (‘closed’ C-Mad2 on binding Mad1 or Cdc20, or ‘open’ O-Mad2 when unbound); interacts with Cdc20 and BubR1/Mad3 to form the MCC, which inhibits the APC; excess Mad2 inhibits the APC/C in many experimental systems. Bub1 Protein kinase; binds to Bub3; requirement for recruiting other checkpoint proteins Syntelic pm32ol.tiff differs depending on system; kinase activity is not required for checkpoint arrest. Mad2 Bub3 Binds to Bub1 and the MCC components; required for Bub1 and BubR1 Journal of Cell Science localisation to the kinetochore. BubR1/Mad3 Binds to Bub3; interacts with Mad2 and Cdc20 to form the MCC; C-terminal Mad2 kinase domain of BubR1 is activated by CENP-E; yeast Mad3, the functional equivalent of BubR1, lacks the kinase domain. Unattached Mps1/Mph1 Protein kinase; phosphorylates Mad1 in vitro; excess activates the checkpoint; required for recruitment of Mad1, Mad2 and CENP-E to the kinetochore. Other proteins required for checkpoint function 0 CENP-E Kinesin family member; binds to BubR1; stimulates BubR1 kinase activity; c2 C Cd required for capture and stabilisation of microtubules at the kinetochore; only found in higher eukaryotes. Kinetochore ? Mps1 Kinetochore Kinetochore RZZ complex Complex of Rod, ZW10 and Zwilch, in higher eukaryotes only recruits dynein 1 1 Mps1 O A and Mad1/Mad2 to the kinetochore. 3 u Mps1 3 BubR1 1 r ub Aurora B ub or Cdc20/Slp1 APC/C activator; binding to Mad2 and BubR1/Mad3 forms the MCC, which ub Aurora B O C RZZ B B a ub ub B B B B inhibits APC/C activation. ub3 O d1 C -E BubR1 a B P Aurora B Protein kinase; chromosomal passenger protein; binds INCENP and survivin; in ub3 le 3 O ub3 M N BubR1 E B B O mammals and budding yeast (Ipl1) required for checkpoint response to lack of C ub -E B CENP- tension but not attachment; required for attachment response in fission yeast (Ark1). P O ubu ? EN O C E p31comet Binds specifically to the ‘closed’ conformation of Mad2; excess disrupts checkpoint O 0 signalling; involved in switching off the checkpoint; no yeast orthologues. C c2 (Cmt2) Cd BubR1 0 Dynein/ Minus-end-directed motor that transports Mad2 and BubR1 c2 Microt away from the kinetochore; thought to be required for switching C Cd RZZ dynactin Mad2 conformations 1 off the checkpoint; dynactin complex recruits ‘cargo’ to dynein. 3 MCC p BubR1 C 0 O C M c2 a Downstream of the checkpoint C Cd C d1 1 3 0 p APC/C E3 ubiquitin ligase; targets mitotic regulators for destruction by the proteosome; 2 p31 downstream effector of the checkpoint. MCC p31 C Cdc C Securin Pds1 in budding yeast, Cut2 in fission yeast, PTTG in human; binds and inhibits separase. MCC inactive? Separase Protease; Esp1 in budding yeast; Cut1 in fission yeast; cleaves the cohesin subunit Scc1, which breaks the cohesin ring. Cohesin Protein complex of Scc1, Smc1 and Smc3 that forms a ring around sister chromatids holding them together; cleavage of Scc1 required for sister separation. BubR1 Ub jcs.biologists.org Ub C c20 BubR1 Ub Cd Ub 20 APC/C C Cdc Ub Mad2 APC/C Ub 0 Ub c2 Ub Cd Ub APC/C No tension No microtubule attachment Checkpoint on Checkpoint off Anaphase © Journal of Cell Science 2006 (119, pp. 4139-4142) (See poster insert) 4140 Journal of Cell Science 119 (20) When a chromosome is attached to kinetochores that could then be sensed recruiting the dynamic BubR1/Mad3 and microtubules from opposite poles, by the Mad/Bub machinery. However, Mad2 proteins, which are candidates tension is generated across the sister the response to lack of tension appears to for the inhibitory signal (Howell et al., kinetochores by the pulling forces of the be more complicated than this, and 2004; Shah et al., 2004). Mad2 binds to spindle. Laser ablation of the last Aurora B kinase has additional Cdc20; this interaction is essential for unattached kinetochore relieves the checkpoint roles in some systems (Kallio checkpoint-dependent inhibition of the checkpoint-dependent arrest and the cell et al., 2002; Petersen and Hagan, 2003). APC/C (Hwang et al., 1998; Kim et al., enters anaphase even though the Thus, whether the core checkpoint 1998). In solution, free Mad2 adopts an remaining sister kinetochore is not under simply detects lack of attachment or is open conformation (O-Mad2) but, on tension (Rieder et al., 1995). This also capable of sensing a lack of tension binding to Mad1 or Cdc20, this changes indicates that lack of microtubule remains controversial (Pinsky and to a stable closed conformation (C- attachment elicits the checkpoint Biggins, 2005). Mad2) (De Antoni et al., 2005). Because response. Conversely, kinetochores Mad1 and Cdc20 compete for the same lacking tension because both sisters are binding site on Mad2, it was initially attached to microtubules from the same Anaphase delay thought that kinetochore recruitment pole (syntelic attachment) activate the The downstream target of the spindle stimulated exchange of inactive Mad2 checkpoint even though kinetochore- checkpoint is the anaphase-promoting from Mad1 to an active form that binds microtubule attachments are made, complex/cyclosome (APC/C), a Cdc20. However, this has recently been indicating that lack of tension can be multiprotein E3 ubiquitin ligase that challenged by two new models in which sufficient for checkpoint activation. ubiquitylates a range of cell-cycle kinetochore-bound Mad1–C-Mad2 is a Chemical inhibition of spindle regulators, targeting them for stable complex that acts as a template dynamics, which relieves tension but degradation by the 26S proteasome recruiting O-Mad2, which is then able to does not destroy kinetochore- (Castro et al., 2005). Securin is the key bind Cdc20 (De Antoni et al., 2005; Yu, microtubule attachments, also activates regulator of anaphase onset and a 2006). Consistent with this idea is the the checkpoint (Clute and Pines, 1999; substrate for the APC/C: its destruction finding that Mad1 and a proportion of Skoufias et al., 2001). However, releases separase, which in turn destroys Mad2 are stably localized to the interpreting such experiments is cohesin (the molecular glue holding kinetochore whereas the remaining complicated because microtubule sister chromatids together) and thus Mad2 and a pool of Cdc20 rapidly cycle attachment is stabilised by tension allows chromatids to be pulled to on and off the kinetochore with similar (Nicklas et al., 2001). opposite poles. APC/C activity is dynamics (Howell et al., 2004; Shah et regulated by the accessory proteins al., 2004; Vink et al., 2006). One clear difference between the Cdc20 and Cdh1, which are thought to checkpoint response to lack of tension interact with specific substrates and Although Mad2 is a good in vitro APC/C and that to lack of attachment is the present them to the APC/C for inhibitor, formation of Mad2-Cdc20 is Journal of Cell Science recruitment of the Mad and Bub proteins. ubiquitylation (Peters, 2002). Cdc20 unlikely to be sufficient to inhibit the Mad1 and Mad2 localise to unattached (Slp1 in fission yeast) is required for the APC in vivo.