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Jonathan McLatchie (B.Sc, M.Res) November 2013
Introduction Mitotic division (“M phase”) is the culmination of the eukaryotic cell cycle for somatic cells. Mitotic cell division is divided into six phases. The first is prophase, which is characterized by chromosome condensation (the reorganization of the sister chromatids into compact rodlike structures). Following condensation, assembly of the mitotic spindle apparatus occurs outside the nucleus between the two centrosomes which have duplicated and moved apart to the poles of the cell.
The second stage of mitosis is prometaphase, which is marked by the disintegration of the nuclear envelope. This is followed by metaphase, where sister chromatids are attached to opposite spindle poles by microtubules bound to protein complexes called kinetochores. In animal cells, 10-40 microtubule-binding sites are associated with any one kinetochore. In yeast, each kinetochore contains only one attachment site. At this point, the chromosomes are seen to be aligned at the cell’s equator (the metaphase plate). The sister chromatids are themselves held together by the protein cohesin.
At anaphase, the sister chromatids separate to form two daughter chromosomes that are pulled towards opposite poles of the spindle. Microtubules bound to kinetochores, as well as the centrosome, are reeled in towards the cell’s periphery by specialized dynein motor proteins that ‘walk’ towards the minus end of the microtubule but are held stationary by cargo-binding domains that are anchored to the cell cortex.
The next phase in the cycle is telophase, the stage at which the daughter chromosomes decondense at the spindle poles and a new nuclear envelope is assembled. A contractile ring is then formed, marking the final stage of the process -- cytokinesis. The contractile ring is comprised of actin and myosin filaments. The cell thus differentiates to form two new daughter cells, each with a nucleus containing a complete and identical set of chromosomes.
The consequences of improper attachment can be catastrophic, with segregation of two chromosome copies to a single daughter cell. The spindle assembly checkpoint pathway is responsible for inhibiting progression of mitosis from metaphase to anaphase until each of
the sister chromatids has become correctly bi-oriented and securely associated with the mitotic spindle.
Progression from metaphase to anaphase is mediated by the anaphase promoting complex (APC), an E3 ubiquitin ligase. When bound to a protein, Cdc20, the APC functions to ubiquitinate securin (a protein that prevents the cleavage of cohesin by the enzyme separase), as well as the S and M cyclins, thereby targeting them for destruction (1-2). The APC is phosphorylated by cyclin dependent kinases (Cdks), thus rendering it able to bind to Cdc20 and form the APCCdc20 complex. The APCCdc20 complex is autoinhibitory, since destruction of Cdks results in a decreased rate of APC phosphorylation and, as a consequence, binding of Cdc20.
Microtubule attachment to kinetochores during prometaphase is governed by a stochastic “search and capture” mechanism (3-5). The property of dynamic instability facilitates the process by which microtubules ‘search’ for kinetochore attachment sites. When a microtubule encounters a kinetochore, the kinetochore is ‘captured’ by means of side-on attachment. The sister chromatids are subsequently positioned at one of the poles of the cell, where more microtubules become attached. After the kinetochore becomes associated with a microtubule from the other pole, the chromosomes move to the equator.
This checkpoint pathway relies on a specialized mechanism for monitoring the security of kinetochore-microtubule attachment (6). In the case of improper attachment, the kinetochore sends out a signal – the wait anaphase signal – that inhibits activation of APCCdc20, thereby arresting metaphase-to-anaphase progression.
The purpose of this paper is to review the elegant molecular mechanisms that underlie the spindle assembly checkpoint and discuss the implications of its dysfunction.
Monitoring Spindle-Kinetochore Attachment The precise mechanism by which the spindle checkpoint system detects improper chromatid bi-orientation has not been fully elucidated. Two main hypotheses have been proposed, each with its own supporting data (7). One proposal suggests that the system monitors the
level of tension at the kinetochore (8-9). Another hypothesis is that the system detects attachment of the ends of the microtubules to the kinetochore (10). The spindle assembly checkpoint pathway most likely uses a combination of those two mechanisms.
The importance of tension sensing in the spindle assembly checkpoint was first examined in insect spermatocytes, using a micromanipulation needle to apply tension to an improperly associated chromosome. Tension resulted in the commencement of anaphase in 56 minutes, whereas it was delayed by 5-6 hours in the absence of tension (8).
Aurora kinase B plays a crucial role in tension sensing, and its inhibition results in an accumulation of improperly attached kinetochores (11-15). Aurora kinase B is believed to induce the inhibitory signal that destabilizes kinetochore-microtubule attachments by phosphorylating components of the kinetochore’s microtubule attachmen t site, including the mammalian histone-H3 variant centromere protein A (CENP-A) at serine 7 (16-17). Aurora kinase B is itself recruited to the centromere by phosphorylation of CENP-A at the same residue by Aurora kinase A (18). When the function of Aurora kinase B is inhibited, one also observes a decrease in concentration of checkpoint components BubR1, Mad2 and CENP-E, and also an inability of BubR1 to rebind to the kinetochore following a decrease in tension at the centromere (19). Aurora kinase B is inactivated only after correct biorientation has occurred.
The role of microtubule attachment is demonstrated by the activity of checkpoint proteins at the kinetochore. For instance, Mad2 is present on unattached kinetochores during prometaphase, but is removed from the kinetochores as they become associated with the spindle (10). Moreover, when mammalian cells are treated with low concentrations of taxol and other microtubule-targeting drugs (thereby removing tension but retaining microtubule-kinetochore attachment), the onset of anaphase is significantly delayed (10,20).
Generating the Wait Anaphase Signal For wild-type yeast cells a spindle defect delays mitotic progression. The molecular components of the spindle assembly checkpoint pathway were first discovered in budding
yeast treated with the microtubule-destabilizing drug benomyl (21-22). The checkpoint components identified in these screens are indispensable in yeast for the spindle checkpoint. These proteins include Mad1, Mad2, Mad3, Bub1 and Bub3. Mad2 mutant cells continue to divide at a normal rate. The spindle defect, however, results in improper segregation of chromosomes and the consequences of this are inevitably lethal. Table 1 shows the key spindle checkpoint components in budding yeast and metazoans, and their respective roles.
Table 1: Spindle Checkpoint Components Metazoans Mad1 Mad2 BubR1 Bub1 Bub3 Mps1 CENP-E Zw10 Rod Budding Yeast Mad1 Mad2 Mad3 Bub1 Bub3 Mps1 No homologues known No homologues known No homologues known Function Binds to Mad2 at kinetochore Binds to Mad1 or Cdc20 Binds to Cdc20 and Bub3 Binds Bub3; serine/threonineprotein kinase Binds Bub1 and BubR1 Protein kinase Binds BubR1; kinase-7 motor protein Complexes with rod Complexes with Zw10
The wait anaphase signal functions by high-affinity binding of checkpoint component Mad2 to Cdc20, thereby inhibiting the APCCdc20 complex and preventing the ubiquitination of securin. Progression to anaphase can be blocked by even a single unattached kinetochore, and it is thought that unattached kinetochores catalyse a conformational change in Mad2, thereby allowing it to bind to Cdc20. In support of this hypothesis is the observation that Mad2 is found at some level at unattached kinetochores and appears to rapidly associate and dissociate with the kinetochore.
During prometaphase, a portion of Mad2 is bound to checkpoint component Mad1. Cdc20 is bound and sequestered by a separate portion of Mad2. Mad2 can be bound to only one of those proteins at a time, since it uses the same site to bind Mad1 and Cdc20. Mad2 has been shown to adopt two distinct natively folded states (23-26). Mad2 adopts a closed conformation (C-Mad2) when bound to one of its partners, Mad1 or Cdc20. In this state, the
carboxy-terminus of Mad2 closes around its binding partner and has been described as a “safety belt” (23). When Mad2 is not bound to Mad1 or Cdc20, it exists in an open conformation (O-Mad2). In this state, the safety belt is held against the side of Mad2, leading to an inability to efficiently bind Mad1 or Cdc20 until the safety belt has been loosened by a conformational change.
What facilitates this change in Mad2 conformation, thereby generating the wait anaphase signal? One proposed model suggests that the formation of Mad2-Cdc20 complexes at incorrectly attached kinetochores is catalysed by Mad2-Mad1 complexes. This hypothesis is supported by the fact that C-Mad2 can form a dimer with O-Mad2, initiate a conformational change that loosens the safety belt, and thereby promote its binding to Cdc20. The newly formed C-Mad2-Cdc20 complex is subsequently released from the kinetochore and, in a remarkable positive feedback loop, catalyses the synthesis of more C-Mad2-Cdc20 complexes by interaction with free O-Mad2 proteins. The C-Mad2-Mad1 complex remains at the kinetochore and repeats the reaction cycle.
A number of other checkpoint components are also involved in inhibition of the APC. Inhibitory complexes consisting of Cdc20 and BubR1 (the mammalian homologue of the yeast protein Mad3) and Bub3 are produced by unattached kinetochores. Together, the Mad2-Cdc20 and BubR1-Bub3-Cdc20 complexes suppress the activation of the APC. Interestingly, the ubiquitination by the APCCdc20 of S-phase cyclin A in prometaphase is not blocked by these inhibitory complexes (27). The explanation for this is unclear, although one possibility is that cyclin A complexes with Cdc20 and competes with spindle checkpoint proteins for binding (27).
Turning Off the Spindle Assembly Checkpoint Pathway Once all of the sister chromatid pairs have been properly bi-oriented on the mitotic spindle, the APCCdc20 is no longer inhibited, and facilitates the ubiquitin-mediated destruction of securin and M-cyclin. What mechanisms ensure that the spindle assembly checkpoint is turned off following proper bi-orientation of sister chromatids? Various checkpoint silencing pathways exist (28). In metazoans, checkpoint components are transported away from kinetochores along microtubules towards the spindle poles in an ATP-dependent manner by
cytoplasmic dynein-dynactin motor complexes (29-30). This process is known as “stripping”. When dynein is inhibited, the removal of Mad1 and Mad2 from the kinetochore is prevented (29). Indeed, when Mad1 is artificially tethered to correctly attached kinetochores, the onset of anaphase is delayed (31). Required for recruitment of dynein to kinetochores are the proteins Spindly and RZZ (rough deal, zeste white 10, zwilch). In cases of Spindly motif mutants that are unable to bind dynein, dynein is not recruited to the kinetochore (32). In such cases, however, the checkpoint is silenced by a second pathway. A further protein, called p31comet (formerly known as CMT2), has also been associated with checkpoint silencing (33-35). By structural mimicry of Mad2, p31comet is able to bind to Mad2 at the dimerization interface, thereby inhibiting its activity. Another protein that has been shown to be involved in checkpoint silencing by dephosphorylating checkpoint components is protein phosphatase 1 (PP1) (36-37).
The Consequences of Checkpoint Dysfunction Checkpoint dysfunction can lead to discrepancies in chromosome number (aneuploidy), the consequences of which include tumorigenesis and Down’s syndrome (38-39). When spindle checkpoint signalling is reduced in mouse models, a rise in cancer development is observed (40). The importance and significance of mutations in spindle checkpoint genes are not entirely clear, since spindle checkpoint mutants are relatively infrequent in human tumours, and colon cells exhibiting chromosomal instability appear to typically possess a fully functional spindle checkpoint (41). More commonly mutated in such cells is the APC gene (42-43). Mutations affecting checkpoint genes is not the primary mechanism of checkpoint impairment. A more frequent cause of aneuploidy is alteration in transcriptional regulation resulting in changes in checkpoint protein levels. Indeed, studies of mice with decreased concentrations of BUBR1, BUB3 and Mad2 manifest a greatly elevated incidence of aneuploid fibroblasts (44-47).
Biallelic mutations in BUB1B (which encodes checkpoint component BUBR1) have been shown to accompany mosaic variegated aneuploidy, an extremely rare condition that predisposes its subject to mitotic non-disjunction, resulting in a significant rise in the
prevalence of aneuploid cells (often greater than 25%), leading to a high incidence of childhood cancers (48).
Conclusion The spindle assembly checkpoint pathway is an elegantly engineered surveillance system for protecting the cell from the adverse consequences of improper kinetochore-microtubule attachment. Proper attachment of kinetochores to microtubules is monitored by tensionsensing and by detection of attachment of the ends of the microtubules to the kinetochores. Even a single unattached kinetochore is sufficient to trigger the wait anaphase signal, which inhibits activation of the APC that drives entry into anaphase. Impairment of the spindle assembly checkpoint pathway can result in aneuploidy, a contributor to cancer and developmental abnormalities such as Down’s syndrome.
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