Hong Hwa Lim

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.

Cdc20 is essential for the cyclosome-mediated proteolysis of both Pds1 and Clb2 during M phase in budding yeast

Chromosome separation during the cell-cycle transition from metaphase to anaphase requires the proteolytic destruction of anaphase inhibitors such as Pds1 [1-3]. Proteolysis of Pds1 is mediated by a ubiquitin-protein ligase, the anaphase-promoting complex (APC) or cyclosome [4,5]. The APC is also necessary for the ubiquitin-dependent degradation of mitotic cyclins in late telophase as cells exit mitosis [6-9]. Although phosphorylation seems to be involved [10], it is not clear what activates the APC at the onset of anaphase. In Saccharomyces cerevisiae, chromosome segregation also requires the CDC20 gene, whose product contains WD40 repeats [11,12]. We have investigated the functional relationship between the APC and the Cdc20 protein. We present evidence that strongly suggests that Cdc20 is an essential regulator of APC-dependent proteolysis such that in the absence of Cdc20, cells are unable to degrade either Pds1 at the onset of anaphase or the mitotic cyclin Clb2 during telophase. This notion is consistent with our observations that Cdc20 is localized in the nucleus and co-immunoprecipitates with an APC component, Cdc23.

Inactivation of Cdh1 by synergistic action of Cdk1 and polo kinase is necessary for proper assembly of the mitotic spindle

Separation of duplicated centrosomes (spindle-pole bodies or SPBs in yeast) is a crucial step in the biogenesis of the mitotic spindle. In vertebrates, centrosome separation requires the BimC family kinesin Eg5 and the activities of Cdk1 and polo kinase; however, the roles of these kinases are not fully understood. In Saccharomyces cerevisiae, SPB separation also requires activated Cdk1 and the plus-end kinesins Cin8 (homologous to vertebrate Eg5) and Kip1. Here we report that polo kinase has a role in the separation of SPBs. We show that adequate accumulation of Cin8 and Kip1 requires inactivation of the anaphase-promoting complex-activator Cdh1 through sequential phosphorylation by Cdk1 and polo kinase. In this process, Cdk1 functions as a priming kinase in that Cdk1-mediated phosphorylation creates a binding site for polo kinase,which further phosphorylates Cdh1. Thus, Cdh1 inactivation through the synergistic action of Cdk1 and polo kinase provides a new model for inactivation of cell-cycle effectors.

Regulation of centrosome separation in yeast and vertebrates: common threads

The assembly of a bipolar spindle is crucial for symmetric partitioning of duplicated chromosomes during cell division. Centrosomes (spindle pole body [SPB] in yeast) constitute the two poles of this bipolar structure and serve as microtubule nucleation centers. A eukaryotic cell enters the division cycle with one centrosome and duplicates it before spindle formation. A proteinaceous link keeps duplicated centrosomes together until it is severed at onset of mitosis, enabling centrosomes to migrate away from each other and assemble a characteristic mitotic spindle. Hence, centrosome separation is crucial in assembly of a bipolar spindle. Whereas centrosome (or SPB) duplication has been characterized in some detail, the separation process is less well understood. Here, we review recent studies that uncover new players and provide a greater understanding of the regulation of centrosome (or SPB) separation.

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.

DNA Damage Checkpoint Maintains Cdh1 in an Active State to Inhibit Anaphase Progression

DNA damage checkpoint prevents segregation of damaged chromosomes by imposing cell-cycle arrest. In budding yeast, Mec1, Chk1, and Rad53 (homologous to human ATM/ATR, Chk1, and Chk2 kinases, respectively) are among the main effectors of this pathway. The DNA damage checkpoint is thought to inhibit chromosome segregation by preventing separase-mediated cleavage of cohesins. Here, we describe a regulatory network that prevents segregation of damaged chromosomes by restricting spindle elongation and acts in parallel with inhibition of cohesin cleavage. This control circuit involves Rad53, polo kinase, the anaphase-promoting complex activator Cdh1, and the bimC kinesin family proteins Cin8 and Kip1. The inhibition of polo kinase by Rad53-dependent phosphorylation prevents it from inactivating Cdh1. As a result, Cdh1 remains in a partially active state and limits Cin8 and Kip1 accumulation, thereby restraining spindle elongation. Hence, the DNA damage checkpoint suppresses both cohesin cleavage and spindle elongation to preserve chromosome stability.

Tome-1, wee1, and the Onset of Mitosis: Coupled Destruction for Timely Entry

In a recent issue of Cell, Ayad et al. (2003) report the identification of a novel F box protein Tome-1, which mediates the destruction of mitosis-inhibitory kinase wee1 via E3 ligase SCF. Tome-1 itself is targeted for degradation by APC in G1. This synergy of destructive action by APC and SCF has important implications for timely entry into mitosis.

Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo.

Chromatin organization is critical to the regulation of most cellular activities. Direct three-dimensional visualization by electron cryotomography shows that budding yeast chromatin is best explained by a polymer melt interspersed with small nucleosome clusters, with no evidence of 30-nm fibers or condensed chromosomes in G1 or mitosis.

Cdk1 promotes kinetochore bi-orientation and regulates Cdc20 expression during recovery from spindle checkpoint arrest.

The spindle assembly checkpoint (SAC), an evolutionarily conserved surveillance pathway, prevents chromosome segregation in response to conditions that disrupt the kinetochore‐microtubule attachment. Removal of the checkpoint‐activating stimulus initiates recovery during which spindle integrity is restored, kinetochores become bi‐oriented, and cells initiate anaphase. Whether recovery ensues passively after the removal of checkpoint stimulus, or requires mediation by specific effectors remains uncertain. Here, we report two unrecognized functions of yeast Cdk1 required for efficient recovery from SAC‐induced arrest. We show that Cdk1 promotes kinetochore bi‐orientation during recovery by restraining premature spindle elongation thereby extinguishing SAC signalling. Moreover, Cdk1 is essential for sustaining the expression of Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) required for anaphase progression. We suggest a model in which Cdk1 activity promotes recovery from SAC‐induced mitotic arrest by regulating bi‐orientation and APC/C activity. Our findings provide fresh insights into the regulation of mitosis and have implications for the therapeutic efficacy of anti‐mitotic drugs.

Deficiency of centromere-associated protein Slk19 causes premature nuclear migration and loss of centromeric elasticity

The cohesin complex prevents premature segregation of duplicated chromosomes by providing resistance to the pole-ward pull by spindle microtubules. The centromeric region (or sister kinetochores) bears the majority of this force and undergoes transient separation prior to anaphase, indicative of its elastic nature. A cysteine protease, separase, cleaves the cohesin subunit Scc1 and dissolves cohesion between sister chromatids, initiating their separation. Separase also cleaves the kinetochore protein Slk19 during anaphase. Slk19 has been implicated in stabilization of the mitotic spindle and regulation of mitotic exit, but it is not known what role it plays at the kinetochores. We show that during pre-anaphase arrest, the spindle in slk19Δ cells is excessively dynamic and the nuclei move into mother-daughter junction prematurely. As a result, the chromatin mass undergoes partial division that requires neither anaphase promoting complex (APC) activity nor Scc1 cleavage. Partial division of the chromatin mass is accompanied by the loss of the centromeric regions ability to resist pole-ward pull by the spindle. Slk19 physically associates with Scc1 and this association appears necessary for efficient cleavage of Slk19 by separase. Our results suggest that Slk19 participates in regulating nuclear migration and, in conjunction with cohesin complex, may be involved in the maintenance of centromeric tensile strength to resist the pole-ward pull.