[Frontiers in Bioscience, 5, d50-57, January 1, 2000]
THE CONTROL OF MITOSIS
Mary L. Hixon 1 and Antonio Gualberto 1,2
1Division of Cardiovascular Research, Department of Medicine, St. Elizabeth's Medical Center, Boston, MA 02135, USA, 2Department of Cardiovascular and Metabolic Diseases, Pfizer Central Research Division, Eastern Point Rd., Groton, CT 06340
TABLE OF CONTENTS
A precise coordination of multiple cell cycle events is required to ensure proper mitosis. Chromosome cohesion must be maintained until all chromosomes are attached to opposite poles of the mitotic spindle and aligned at the metaphase plate. At the onset of anaphase, the activity of separins contributes to the release of cohesins from chromosomes, allowing for the segregation of bivalents to opposite spindle poles. Separin activity is blocked by binding to a class of proteins known as securins, whose turnover at the metaphase-to-anaphase transition is triggered by the Anaphase Promoting Complex or cyclosome. The mitotic spindle cell cycle checkpoint coordinates the timing of these events and acts as input mechanism for DNA damage/stress pathways. Failure of this precise network leads to genomic instability and/or cell death.
Mitosis is a complex process which includes nuclear envelope breakdown, chromatin condensation and chromosome segregation. Thus, multiple events must be coordinated in order to ensure the progression of mitosis. Although much is already known about the genetic and biochemical components that constitute the cell cycle machinery at mitosis, how these pathways are coordinated has been largely unknown until recently.
Since 1966, the existence of factors which control mitotic entry has been postulated. The first evidence for such factors came from fusion experiments in P. polycephalum (1). These data were later supported by experiments involving the fusion of mammalian cells arrested at G1, S, or G2 phase of the cell cycle with cells which arrested at mitosis, which results in chromosome condensation (2-4). Biochemical and genetic experiments established that the onset of mitosis is controlled by the activity of the mitosis promoting factor (MPF), a complex consisting of the p34 Cdc2 cyclin-dependent kinase, Cyclin B and associated proteins (5). MPF induces the formation of a mitotic spindle by phosphorylating the components of the mitotic spindle apparatus, including microtubule-based motor proteins (6). In addition, MPF activates NimA (never in mitosis), a kinase that is required for spindle formation (7). After anaphase, the destruction of the mitotic cyclins leads to MPF inactivation, originating spindle disassembly and cytokinesis (8,9).
While the activity and subunit composition of MPF have been extensively investigated, less is known about the molecular events that control the metaphase-to-anaphase transition and the exit from mitosis. However, in the last few years, extensive progress has been achieved in the understanding of these processes. Importantly, recent data indicate that the destruction of sister chromatid cohesion, rather than pulling forces, is responsible for the separation of sister chromatids at the metaphase to anaphase transition (10). Here, we will review the regulatory mechanisms that control chromatid cohesion, the onset of anaphase and the exit from mitosis.
3. 1. Chromatid Cohesion
Chromatid cohesion allows for sister chromatids to remain together from the time of DNA replication to mitosis. Chomatid cohesion also promotes proper microtubule attachment by constraining kinetochores (11,12). Multiple chromosome cohesion proteins have been identified. They include Mcd1(Scc1)p, Scc3p, Smc1p, and Smc3p in budding yeast (13-16) and Mis4 in fission yeast (17). In Xenopus, homologs of Mcd1p, Smc1p, and Smc3p have also been recently identified (18). Smc1p and Smc3p are members of the structural maintenance of chromosomes (SMC) gene family (19). Mcd1p requires Smc1p to associate with chromatin, plays a role in chromosome condensation (13,14), and is homologous to fission yeast Rad21 (20). The process of chromosome condensation; however, is independent from sister chromatid cohesion (18) and is mediated by two additional SMC family members, Smc2p and Smc4p (19,21-23). Importantly, chromosome cohesion varies during mitosis (24). These changes may result from the dissociation of Mcd1p, Smc1p, and Smc3p from the chromosome along the progression of mitosis (18). Another Rad21 homologue, Rec8, seems to play a central in yeast chromosome cohesion during meiosis (25).
In fission yeasts, Mis4, initially identified by its requirement for stable maintenance of minichromosomes (26), was found to be also required for sister chromatid cohesion (17,27), although its function seems to be quite different from cohesin. Temperature-sensitive mis4 mutants are lethal at restrictive temperature and grow at the permissive temperature with loss of minichromosomes. To date, twelve mis genes have been identified. The mis5 gene encodes one of the MCM subunits essential for replication (26,28); whereas, mis6 encodes for a centromere protein essential for the formation of centromere-specific chromatin in the G1/S phase (29). The phenotype of mis6 mutants is similar to that of mis4, however, not only minichromosomes, but also normal chromosomes are missegregated (26). Thus, mis genes appear to play a variety of important roles in maintaining chromosomal dynamics and integrity.
In Drosophila, Mei-S332 is required for the maintenance of sister chromatid cohesion at meiosis (30,31). This protein localizes to centromeres until sister chromatids separate and dissociate from chromosomes at anaphase, suggesting that its release is involved in sister chromatid separation (30). Mutations in the mei-S332 gene lead to early sister chromatid separation and chromosome loss or mis-segregation (32).
In Xenopus, recent work has identified homologs of budding yeast Smc1p,Smc3p, Mcd1p/Scc1p and Rad21p (18). In contrast to yeast, the cohesin complex of frog extracts does not persist throughout mitosis. It dissociates at the onset of mitosis when it is replaced by the condensin complex (18).
3.1.1. Separin, Securin and the onset of anaphase
In addition to structural proteins, the cohesion complex includes also proteins with regulatory function. The dissociation of cohesins from chromatids results in sister chromatid separation and seems to be triggered by proteolytic cleavage of Scc1p. This cleavage depends upon the activity of a separin, Esp1p (figure 1) (33,34). Esp1p is essential for the dissociation of Scc1p from all regions of the chromosome. Furthermore, Scc1p dissociation from chromosomes is accompanied by its cleavage at two specific sites and requires Esp1p (35-37). However, it is not known whether Esp1p is a Scc1p protease or if it activates a protease that cleaves Scc1p.
Figure 1. Model of Separin/Securin control by the APC/C.
Importantly, Esp1p is tighly associated to Pds1p (securin) and this association inhibits Esp1p separin function and the onset of anaphase (36,38,39). The carboxy terminal region of Esp1p has high sequence similarity with the fission yeast cut1 and A. nidulans bimB gene products, which are required for chromosome segregation (40,41). In cut1 mutants, centromeres and most of the chromosome separate normally, but telomeric cohesion remains (41,42). Cut1 also associates with an inhibitor protein, Cut2 (36,41-43).
A vertebrate securin (vSecurin) has recently been identified on the basis of its biochemical analogy to Pds1p and Cut2. The vSecurin protein binds to a vertebrate homolog of Esp1p and Cut1, and is degraded by proteolysis mediated by the cyclosome complex (44). Human securin is identical to the product of the gene called pituitary tumor-transforming gene (PTTG), which is overexpressed in some tumors and exhibits transforming activity in NIH 3T3 cells (44). Furthermore, the addition of a non-degradable form of PTTG to Xenopus egg extracts prevented sister chromatid separation but not exit from mitosis. These parallels demonstrate that both securin and separin functions are well conserved.
3.2. The Anaphase Promoting Complex
Both, the metaphase-to-anaphase transition and the exit from mitosis, are regulated by protein degradation mediated by a specific ubiquitin ligase complex known as the Anaphase-Promoting Complex or Cyclosome (APC/C) (figure 2) (45-49). APC/C targets contain a conserved nine amino acid motif called the destruction box that is required for APC/C-mediated ubiquitination (46, 50-55). During this process, ubiquitin is activated forming a thioester bond with a cysteine residue at the E1 ubiquitin-activating enzyme. Ubiquitin is then transferred to an ubiquitin-conjugating enzyme, called UBC or E2, forming a second thioester. Finally, an isopeptide bond is formed between ubiquitin and a lysine residue at the substrate. More ubiquitin molecules can then be conjugated to those already attached creating a polyubiquitin chain that is targeted for degradation by the 26S proteasome (56). The transfer of ubiquitin from E2 enzymes to substrates requires a third activity, called ubiquitin-protein ligase or E3. Unlike E1 and E2, ubiquitin ligase activity is cell cycle regulated, active from anaphase up to late G1 phase. Fractionation experiments using extracts from clam and Xenopus eggs demonstrated that this ligase activity sedimented as a large particle of 20S. The clam ubiquitin ligase particle was called the cyclosome, the yeast and Xenopus particles were called the anaphase-promoting complex. The composition of APC/C was further investigated by immunopurification. The Xenopus and human particles were found to contain 10 subunits, whereas the budding yeast particle contains 12 subunits. The core APC/C subunits in mammals are Tgs24, Apc2, Cdc16, Cdc27, Apc7, Apc4, Apc5, Cdc23, Apc10, Apc11 and Cdc26 (56).
3.2.1. The Control of APC/C activity
The activity of the APC/C is regulated at multiple levels. WD-repeat regulatory proteins seem to work as adapters that confer substrate specificity to the APC/C (54-57). Importantly, since mitotic cyclin degradation must not take place until cells are ready to exit mitosis, the APC/C that promotes the metabolism of the anaphase inhibitor at the metaphase-to-anaphase transition cannot simultaneously promote the destruction of mitotic cyclins. In budding yeast, at least two different forms of APC/C have been demonstrated. These forms can be distinguished by their association with the WD-repeat regulatory proteins Cdc20p (Cdc20/p55CDC/Fizzy) and Hct1(Cdh1)p (57-60). Cdc20p mediates the degradation of Pds1p (61,62) and the mitotic cyclin Clb3p (63) at the metaphase-to-anaphase transition. On the other hand, Hct1(Cdh1)p promotes the degradation at anaphase of the mitotic cyclin Clb2, the polo-like kinase Cdc5p, and the spindle-associated Ase1p (57,64,65). In addition, Hct1(Cdh1)p associates with the APC/C later in the cell cycle than Cdc20p. Cdc20p levels and association to the APC/C rise and fall at mitotic entry and exit. In contrast, the level of Hct1(Cdh1)p is constant, but its association to the APC/C is observed from anaphase to G1 (49,66). Furthermore, Hct1p/APC/C association requires dephosphorylated Hct1p (49, 67,68). In addition to Cdc20p and Hct1(Cdh1)p, other WD proteins involved in the control of mitosis have been recently identified, including the fission yeast Cdc20p homolog Slp1 and the Hct1(Cdh1)p homolog Srw1 (69-72). The function of these proteins, however, is less understood.
The activity of the APC/C is also regulated by cell cycle-specific phosphorylation (45,46,55,57,58,66,73-78). Reversible phosphorylation by MPF inactivates APC/C activity (45,73,79,80). In addition, Polo-like kinase 1 (Plk1), a mammalian homologue of Drosophila polo and budding yeast Cdc5p, phosphorylates at least three APC/C subunits, Apc1, Apc3, and Apc6, activating the APC/C (figure 2) (69-66,81-83). In contrast, cAMP-dependent protein kinase (PKA) phosphorylates Apc1 and Apc3 (82) suppressing APC/C activity (74,75,82,84). Furthermore, protein phosphatase 1 (PP1) has been found to be required for the activation of APC/C during the metaphase-to-anaphase transition (74). Hct1(Cdh1)p is also dephosphorylated by Cdc14p (49,67,68), which may be itself regulated by Plk1/Cdc5p and the Cdc15p kinase, all of which are essential for mitotic exit (67,85,86). The yeast inhibitor of mitosis, Sic1p, is regulated by similar mechanisms: Cdk1-dependent phosphorylation inhibits its synthesis and stability, and Cdc14-dependent dephosphorylation reverses these effects and leads to Sic1p accumulation (67,87-89).
Finally, the APC/C is inactivated by the mitotic spindle cell cycle checkpoint pathway (78,90-95). The checkpoint proteins Mad1, Mad2, Mad3/Bub1, and Bub3 suppress the activation of APC/C by Cdc20p. Also, Bub2p localizes to the spindle pole body and regulates Hct1(Cdh1)p -dependent APC/C activation (63,96,97). Dephosphorylation of Hct1(Cdh1)p by Cdc14p is controlled by Bub2(Byr4)p and the RENT (regulator of nucleolar silencing and telophase exit) complex (63,85,86).
3.3. The Mitotic Spindle Cell Cycle Checkpoint
The progression through mitosis is monitored by surveillance mechanisms (98). One of these surveillance mechanisms, known as the mitotic spindle cell cycle checkpoint, ensures the proper segregation of chromosomes by establishing the timing of anaphase onset (99) and by monitoring the attachment of microtubules to the kinetochores and the tension exerted on the kinetochores by the microtubules (100,101). Studies in yeast, Drosophila, and Xenopus have identified multiple genes involved in the activity of this checkpoint (102-104). Initially, the MAD (mitotic arrest deficient) and BUB (budding uninhibited by benomyl) genes were identified by a screening for mutants sensitive to microtubule depolymerizing compounds (102,103). It was found that, in response to mitotic spindle depolymerizing agents, mutations in any of these genes (BUB1, BUB2, or BUB3, and MAD1, MAD2, or MAD3 ) resulted in aberrant mitosis and cell death. Another gene, MPS1, is required for spindle pole body duplication and has been shown to be involved in cell cycle checkpoint control (105). MPS1 encodes a protein kinase believed to function in the signalling pathway (106). Hence, the spindle checkpoint detects spindle pole body duplication and defects in microtubule polymerization, microtubule motors or kinetochore components (107).
The recent characterization of some of the MAD and BUB gene products have determined their localization within the cell. For example, Mad2 protein has been shown to localize to kinetochores that are not yet attached to the mitotic spindle (78,90,97,107-109). Similar to its human counterpart, the murine homolog of yeast Bub1p localizes to unattached kinetochores during mitosis (99). Additional genetic and biochemical studies have also shown that Bub1p, Bub3p, and Mps1p act upstream of Mad1p and Mad2p; whereas, Bub2p and Mad3p act either downstream of Mad proteins, or in a parallel pathway (98,101). Both the human and Xenopus homologs of the yeast Mad2p, HMAD2 and XMAD2, have been cloned and have been shown to be required for spindle checkpoint control (108,109). At the present time, however, it is not known if all the Mad and Bub proteins form a complex at the kinetochore. Generally, a normal bipolar attachment at the kinetochore results in dissociation of the Bub1 and Mad2 proteins from the kinetochore. However, if the kinetochore is unattached from the microtubules, the Bub and Mad proteins remain bound to the kinetochore and arrest the cell in metaphase, delaying the activation of the APC/C (110).
Cdc20p-dependent proteolysis appears to be the target of the mitotic spindle cell cycle checkpoint pathway (59,63,78,92,94,97,111). Importantly, some studies have recently identified a physical interaction between Mad2p and the APC/C. Mad2p binds Cdc20p, inhibiting the destruction of cyclins as well as Pds1p (59,63,78,92,94). The checkpoint-mediated inhibition of APC/C has been reconstituted in vitro with purified Mad2p and Cdc20p. Intriguingly, Mad2p exists in two different forms, a tetramer and a monomer. Only the tetramer inhibits the activation of APC/C by Cdc20p in vitro, suggesting that a structural change of Mad2p may be necessary for transducing the checkpoint signal (92). There is also evidence suggesting that Cdc20p is a target of the DNA damage response; overexpression of Cdc20p overrides metaphase arrest caused by DNA damage (94,111). However, evidence is still lacking for a direct regulation of Cdc20p to the DNA damage response pathway.
Mitosis is a complex process which comprises multiple and simultaneous events that require precise regulatory mechanisms. Protein-protein association, protein phosphorylation and proteolysis play a key role in the regulation of the mitotic regulatory machinery. APC/C-dependent proteolysis drives both metaphase-to-anaphase transition and exit from mitosis and is regulated by at least three mechanisms: Activation by Cdc20p and Cdh1p; phosphorylation and dephosphorylation; and inhibition by the mitotic spindle cell cycle checkpoint.
Destruction of sister chromatid cohesion is responsible for sister chromatid separation. Dissociation of cohesin complexes from chromatids is promoted by separins whose activity is blocked by binding to securins. Securin turnover, triggered by the APC/C, determines chromatid separation and anaphase onset. Yet many unanswered questions remain as to the regulation of separin/securins by the APC/C and the localization of the cohesion complex components on the chromosome. Future work should determine the additional players of sister chromatid cohesion during both mitosis and interphase, the localization of these proteins along the chromosome and how their function is regulated by mitotic signals.
It is established that the APC/C is a multisubunit complex that promotes the proteosome-mediated proteolysis of key regulators of mitosis. However, very little is known about the functions of the individual APC/C components and how the function of these components is regulated. Is the activity of the APC/C an all-or-nothing mechanism ? It is also anticipated that new members of this complex will be soon identified in vertebrates.
Superimposed upon this, the mitotic spindle cell cycle checkpoint pathway ensures the order of the entire mitotic process and responds also to DNA damage signals, detecting spindle pole body duplications and defects in microtubule polymerization, microtubule motors or kinetochore components. Mitotic spindle sensors localize at the kinetochore and dissociate from this structure following normal bipolar attachment. In contrast, Bub and Mad proteins remain bound to unattached kinetochores determining growth arrest in metaphase. It is possible that the event sensed by these proteins is the tension generated on the kinetochore by the microtubular attachment to a bipolar spindle and some evidence suggest that both attachment and tension are required for metaphase-to-anaphase progression (112). In addition, is the APC/C the only target of the mitotic spindle checkpoint pathway? Interestingly, recent data indicate that mitotic checkpoint signals downregulate the expression of hCks1 (113), a Cdc2-associated protein that has been implicated in the control of both APC/C and proteosome functions (114-116).
In conclusion, it is evident that the control of mitosis involves genes involved in the regulation of metaphase-anaphase transition, exit from mitosis and cell cycle checkpoint function. These genes interact in intricate pathways to ensure the accurate segregation of the genome. The study of these pathways constitutes an exciting area of research in molecular and cellular biology.
We apologize to colleagues whose work was not cited due to space limitations. We thank the American Heart Association and the Ohio Cancer Research Associates for grant support.
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