Foong May Yeong

Exit from Mitosis in Budding Yeast: Biphasic Inactivation of the Cdc28-Clb2 Mitotic Kinase and the Role of Cdc20

Cdc20, an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae. Here we show that during mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps. The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20. The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1. The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second. Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1.

Exit from mitosis triggers Chs2p transport from the endoplasmic reticulum to mother–daughter neck via the secretory pathway in budding yeast

Budding yeast chitin synthase 2 (Chs2p), which lays down the primary septum, localizes to the mother–daughter neck in telophase. However, the mechanism underlying the timely neck localization of Chs2p is not known. Recently, it was found that a component of the exocyst complex, Sec3p–green fluorescent protein, arrives at the neck upon mitotic exit. It is not clear whether the neck localization of Chs2p, which is a cargo of the exocyst complex, was similarly regulated by mitotic exit. We report that Chs2p was restrained in the endoplasmic reticulum (ER) during metaphase. Furthermore, mitotic exit was sufficient to cause Chs2p neck localization specifically by triggering the Sec12p-dependent transport of Chs2p out of the ER. Chs2p was “forced” prematurely to the neck by mitotic kinase inactivation at metaphase, with chitin deposition occurring between mother and daughter cells. The dependence of Chs2p exit from the ER followed by its transport to the neck upon mitotic exit ensures that septum formation occurs only after the completion of mitotic events.

Dependence of Chs2 ER export on dephosphorylation by cytoplasmic Cdc14 ensures that septum formation follows mitosis

Sequestration of Cdc14 from the cytoplasm ensures Chs2 ER retention after MEN activation. The interdependence of chromosome segregation, MEN activation, decrease in mitotic CDK activity, and Cdc14 dispersal provides an effective mechanism for cells to order late mitotic events.

Safeguarding Entry into Mitosis: the Antephase Checkpoint

ABSTRACT Maintenance of genomic stability is needed for cells to survive many rounds of division throughout their lifetime. Key to the proper inheritance of intact genome is the tight temporal and spatial coordination of cell cycle events. Moreover, checkpoints are present that function to monitor the proper execution of cell cycle processes. For instance, the DNA damage and spindle assembly checkpoints ensure genomic integrity by delaying cell cycle progression in the presence of DNA or spindle damage, respectively. A checkpoint that has recently been gaining attention is the antephase checkpoint that acts to prevent cells from entering mitosis in response to a range of stress agents. We review here what is known about the pathway that monitors the status of the cells at the brink of entry into mitosis when cells are exposed to insults that threaten the proper inheritance of chromosomes. We highlight issues which are unresolved in terms of our understanding of the antephase checkpoint and provide some perspectives on what lies ahead in the understanding of how the checkpoint functions.

Severing all ties between mother and daughter: cell separation in budding yeast

At the end of nuclear division in the budding yeast, acto‐myosin ring contraction and cytokinesis occur between mother and daughter cells. This is followed by cell separation, after which mother and daughter cells go their separate ways. While cell separation may be the last event that takes place between the two cells, it is nonetheless under tight regulation which ensures that both cells are viable upon separation. It is becoming increasingly clear that the components of the cell separation machinery are controlled at various levels, including the temporal and spatial regulation of the genes encoding for the components and the specific localization of the components to the neck. In addition, these regulatory controls are co‐ordinated with exit from mitosis, thereby placing a mechanistic link between the end of mitosis and cell separation. More importantly, the success of the cell separation event is contingent upon the presence of a trilaminar septum, whose assembly is dependent on a host of proteins which localize to the neck over the span of one cell division cycle.

Retention of Chs2p in the ER requires N-terminal CDK1-phosphorylation sites

In budding yeast, the secretory pathway is constitutively transporting cargoes such as invertase and α-factor throughout the cell division cycle. However, chitin synthase 2 (Chs2p), another cargo of the secretory pathway, is retained at the endoplasmic reticulum (ER) during mitosis when the mitotic kinase activity is high. Chs2p is exported from the ER to the mother-daughter neck only upon mitotic kinase destruction, indicating that the mitotic kinase activity is critical for the ER retention of Chs2p. However, a key question is whether the mitotic kinase acts directly upon Chs2p to prevent its ER export. We report here that mutation of Ser residues to Glu at 4 perfect CDK1-phosphorylation sites at the N-terminus of Chs2p leads to its retention in the ER when the mitotic kinase activity is absent. Conversely, Ser-to-Ala mutations result in the loss of Chs2p ER retention even when mitotic kinase activity is high. The mere over-expression of the non-destructible form of the mitotic cyclin in G1 cells can confine the wild-type Chs2p but not the Ser-to-Ala mutant in the ER. Furthermore, over-expression of the Ser-to-Ala mutant kills cells. Time-lapsed imaging revealed that Chs2p is exported from the ER rapidly and synchronously to the Golgi upon metaphase release. Our data indicate that direct phosphorylation of Chs2p by the mitotic CDK1 helps restrain it in the ER during mitosis to prevent its rapid export in an untimely manner until after sister chromatid occurs and mitotic exit executed.

Early Expressed Clb Proteins Allow Accumulation of Mitotic Cyclin by Inactivating Proteolytic Machinery during S Phase

ABSTRACT Periodic accumulation and destruction of mitotic cyclins are important for the initiation and termination of M phase. It is known that both APCCdc20 and APCHct1 collaborate to destroy mitotic cyclins during M phase. Here we show that this relationship between anaphase-promoting complex (APC) and Clb proteins is reversed in S phase such that the early Clb kinases (Clb3, Clb4, and Clb5 kinases) inactivate APCHct1 to allow Clb2 accumulation. This alternating antagonism between APC and Clb proteins during S and M phases constitutes an oscillatory system that generates undulations in the levels of mitotic cyclins.

CHFR: a key checkpoint component implicated in a wide range of cancers

CHFR (Checkpoint with Forkhead-associated and RING finger domains) has been implicated in a checkpoint regulating entry into mitosis. However, the details underlying its roles and regulation are unclear due to conflicting lines of evidence supporting different notions of its functions. We provide here an overview of how CHFR is thought to contribute towards regulating mitotic entry and present possible explanations for contradictory observations published on the functions and regulation of CHFR. Furthermore, we survey key data showing correlations between promoter hypermethylation or down-regulation of CHFR and cancers, with a view on the likely reasons why different extents of correlations have been reported. Lastly, we explore the possibilities of exploiting CHFR promoter hypermethylation status in diagnostics and therapeutics for cancer patients. With keen interest currently focused on the association between hypermethylation of CHFR and cancers, details of how CHFR functions require further study to reveal how its absence might possibly contribute to tumorigenesis.

Anaphase-Promoting Complex in Caenorhabditis elegans

Progression through critical events in cell division relies to a large extent on the destruction of key effectors of the cell cycle. An example can be seen in the regulation of the major cell cycle effector, the cyclin-dependent kinases (CDKs). CDKs depend upon the binding of their activators, the cyclins, to drive specific events at particular stages in the cell division cycle. As different cyclins are synthesized at different phases of the cell cycle, the CDK, when associated with cyclins, essentially exhibits distinct activities in each phase of the cell cycle. The destruction of cell cycle stage-specific cyclins therefore plays an important role in limiting the activities of the CDKs at the end of each stage so that the CDKs can associate with yet other cyclins to drive progression of the cell cycle (reviewed in reference 45). Cyclins were initially discovered as proteins whose levels oscillated in a highly specific manner in sea urchin Arbacia punctulata embryos undergoing successive rounds of cell division (12). Initial hints that the cyclical nature of the cyclins is important for the regulation of the cell cycle came from the observation that destruction of mitotic cyclins, leading to a decrease in mitotic kinase activities, is required for the exit from mitosis (47). Further analyses led to the understanding that ubiquitination is key to cyclin destruction (19, 28). An E3 ubiquitin-ligase known as the anaphase-promoting complex (APC) was subsequently purified from extracts of the clam Spisula solidissima (29, 64) and African clawed frog Xenopus laevis (33) and shown to be responsible for the ubiquitination of mitotic cyclins. In the process of ubiquitination, the E3 ligase adds a chain of ubiquitin, a 76-residue polypeptide, to proteins destined for destruction. The proteins marked with a ubiquitin chain are recognized as substrates by the 26S proteosome and duly destroyed (reviewed in references 26 and 70). Studies in the budding yeast Saccharomyces cerevisiae revealed that mutants of the APC subunits such as cdc16 and cdc23 were defective for anaphase progression (31), indicating that the APC is required for mediating destruction of regulators for metaphase to anaphase transition in addition to the ubiquitination of mitotic cyclins. To date, the evolutionarily conserved APC (reviewed in references 25 and 54) has been shown to consist of at least 11 subunits (Table ​(Table1),1), and its activity is cell cycle regulated such that it is active in mitosis (reviewed in reference 50). Activation of the APC activity depends upon the association of WD-40 repeat proteins known as Cdc20/Fizzy and its homologue, Cdh1/Fizzy-related (reviewed in references 25, 50, 54 and 78). These activators have been found to be highly conserved across species (reviewed in references 25 and 54). TABLE 1. Orthologues of APC subunits in C. elegans While the functions of each individual APC subunit remain unclear, a comprehensive picture of the roles of the APC in mitosis has emerged through several important studies in budding yeast. The APCCdc20 and APCHct1 complexes have essentially been shown to be required for two main transitions in mitosis, metaphase-to-anaphase transition and exit from mitosis. In metaphase, sister chromatids are held together by a cohesin complex presently known to consist of at least four subunits, Smc1, Smc3, Scc1/Mcd1/Rad21, and Scc3 (reviewed in reference 48) (Fig. ​(Fig.1).1). In order that sister chromatids separate, Scc1 has to be cleaved by separase, a cysteine protease belonging to the CD clan (68, 69). Separase, however, is normally held inactive during metaphase through its association with the anaphase inhibitor securin. APCCdc20 functions to promote sister chromatid separation by ubiquitination of securin and in so doing causes its destruction (reviewed in reference 48) (Fig. ​(Fig.1).1). The liberated separase is then free to cleave Scc1, leading to loss of sister chromatid cohesion, thereby allowing chromatids to be pulled apart by the spindle microtubules (Fig. ​(Fig.1).1). Cdc20 is the target of the spindle assembly checkpoint, which is activated in the presence of spindle defects. The spindle assembly checkpoint components such as Mad1, Mad2, Mad3, Bub1, Bub3, and Mps1 are involved in a pathway that serves to sequester Cdc20, thereby preventing the onset of anaphase, when spindle microtubules are disrupted (reviewed in reference 77). FIG. 1. Diagrammatic representation of sister chromatid separation. Sister chromatids are held together by cohesin complexes (short black bars) during metaphase. APCCdc20 ubiquitination of securin (red) leads to liberation of separase (blue). Separase cleaves ... Homologues of the APC subunits, securin, separase, and cohesin subunits and even the spindle assembly checkpoint components have been found in different organisms (reviewed in reference 48), a strong indication that the pathway leading to sister chromatid separation is perhaps rather well conserved. APCCdc20 also triggers the destruction of S and M phase cyclins to initiate exit from mitosis so that cells can enter a new round of cell division. The initial lowering of the mitotic kinase activities by APCCdc20 (3, 30, 73, 76) allows APCHct1 to be activated by the mitotic exit network (MEN), comprising components such as Tem1 (GTPase), Cdc15 (kinase), Dbf2 (kinase), and Cdc14 (phosphatase) (reviewed in reference 46). The MEN pathway is the target for another type of spindle checkpoint, the spindle position checkpoint. This checkpoint depends upon Bub2 and monitors the orientation and position of the spindle apparatus. In the event that the spindle apparatus is misoriented, Bub2 acts through the MEN pathway to delay mitotic exit via Hct1 until the spindle apparatus is properly oriented (reviewed in reference 40). It has also been shown in budding yeast that homologous chromosome separation during meiosis takes place only in the presence of a functional APC. As in sister chromatid segregation, the APC promotes the ubiquitination of securin in meiosis I and II (59). Degradation of securin by the 26S proteosome frees separase (4, 69), which cleaves Rec8, a meiosis-specific cohesin subunit (37) that shows homology to Scc1 (see above). During meiosis I, the Rec8 molecules localized to the longitudinal arms are destroyed by separase, leading to homologous chromosome disjunction, while Rec8 in the proximity of the centromeres is protected from proteolysis until meiosis II (4, 37). The direct involvement of separase in meiosis II has yet to be confirmed. The sequential destruction in meiosis of Rec8 homologues also occurs in other organisms, such as Schizosaccharomyces pombe (74), Mus musculus (39), and Arabidopsis thaliana (5). Insofar as our understanding of APC function in cell division is concerned, the budding yeast has been instrumental in providing the initial insights. With the identification of homologues of the APC subunits and activators and the other components of chromosome separation and cohesion pathways (reviewed in reference 48), it would be interesting to establish the extent of conservation in the functions of the APC in different organisms. Furthermore, the question now arises as to whether the APC, which is essential in cell division, also contributes to other processes in multicellular organisms. Indeed, recent reviews on the APC have alluded to the possibility that the APC performs functions in cellular processes other than mitosis and meiosis (25, 54). Support for additional roles of the APC comes from studies showing that Hct1/Cdh1 and APC subunits can be detected in postmitotic murine neuronal cells (18) and that multiple copies of Hct1/Cdh1 genes exist in chickens (72). Interestingly, however, work by various groups on the APC in Caenorhabditis elegans in the past few years has not only implicated APC subunits in meiosis and mitosis; more significantly, the data indicate that the functions of the APC in meiotic and mitotic divisions have important consequences in the development of the worm (Table ​(Table1).1). This would suggest that at least in C. elegans, additional roles of the APC, such as in development, are a result of its contribution to proper cell division. Although C. elegans has not been the major organism of choice for studying the APC, it has nonetheless lent itself as a useful system for a better understanding of APC function for several reasons. First, as the APC subunits, Cdc20/Fizzy and Hct1/Fizzy-related have been found in C. elegans (Table ​(Table1),1), comparisons can clearly be made to establish the similarities and differences in APC function across different species. Second, an advantage of using C. elegans is the availability of traditional genetic techniques which make the generation of APC mutant worms for functional studies relatively easy. Also, as a reverse genetics approach, the newly established double-stranded RNA interference (dsRNAi) method provides a powerful and convenient tool for targeting genes in knockdown experiments in C. elegans (15). Furthermore, the well-characterized behavior of the homologous chromosomes (five pairs of autosomes and one pair of sex chromosomes in the hermaphrodite) in worm oocytes has been useful for examining meiotic progression, while the single-celled embryo has characteristics which allow cytological observations of mitotic division processes such as spindles, nucleus, and centrosomes by Nomarski optics. Finally, with the complete documentation of the entire cell lineage (65) and an anatomy that has been well studied (reviewed in reference 38), C. elegans can potentially serve as an important model for investigating the role of the APC in development. This review takes a look at what is currently known about the contributions of APC in cell division as well as how the functions of the APC in cell division impinge upon the development of C. elegans.

Multi‐step down‐regulation of the secretory pathway in mitosis: A fresh perspective on protein trafficking

The secretory pathway delivers proteins synthesized at the rough endoplasmic reticulum (RER) to various subcellular locations via the Golgi apparatus. Currently, efforts are focused on understanding the molecular machineries driving individual processes at the RER and Golgi that package, modify and transport proteins. However, studies are routinely performed using non‐dividing cells. This obscures the critical issue of how the secretory pathway is affected by cell division. Indeed, several studies have indicated that protein trafficking is down‐regulated during mitosis. Moreover, the RER and Golgi apparatus exhibit gross reorganization in mitosis. Here I provide a relatively neglected perspective of how the mitotic cyclin‐dependent kinase (CDK1) could regulate various stages of the secretory pathway. I highlight several aspects of the mitotic control of protein trafficking that remain unresolved and suggest that further studies on how the mitotic CDK1 influences the secretory pathway are necessary to obtain a deeper understanding of protein transport.