Chromosome Segregation

Aneuploidy, i.e. missing or supernumerary chromosomes in the cell nucleus, is a hallmark of malignant tumour progression. A large number of genes that orchestrate faithful chromosome segregation during mitotic cell divisions are tumour suppressors or turn into potent oncogenes if misregulated. The aim of the Chromosome Segregation Laboratory is to investigate cellular mechanisms that safeguard accurate chromosome segregation. In particular, we are investigating the contribution of structural chromosomal proteins to sister chromatid cohesion and chromosome condensation, essential processes that ensure faithful segregation of the centimetre-long chromosomal DNA molecules within micrometre-sized cells. A second topic of our research is the regulation of ordered mitotic progression by the cell division cycle machinery.

The irreversibility of cell cycle transitions

The eukaryotic cell division cycle comprises an ordered series of events, orchestrated by the activity of cyclin-dependent kinases (Cdks). In every cell cycle, chromosomes are first replicated during S-phase and later segregated during mitosis. The unidirectionality of cell cycle transitions is fundamental for successful completion of this cycle. For example, the transition in G1 phase to enter S-phase must be taken only once every cell cycle to avoid deleterious over-replication of the genome. It is thought that the irrevocable nature of proteolytic degradation of key cell cycle regulators makes cell cycle transitions irreversible, thereby enforcing directionality. At the G1/S transition, Cdk inhibitory proteins are proteolysed to allow entry into the cell cycle.

Here, we have studied the irreversibility of mitotic exit. After completion of chromosome segregation during mitosis, Cdk activity is downregulated to promote mitotic exit, i.e. return of cells to G1. After this, cells must not return to mitosis before going through S-phase first. An attempted second round of chromosome segregation would lead to severe aneuploidy. Mitotic exit is driven by ubiquitinmediated degradation of mitotic cyclins under control of the anaphase promoting complex (APC), a multi-subunit ubiquitin ligase. Mitotic cyclins are initially targeted for degradation by the APC in association with its activating subunit Cdc20 (APC^Cdc20). Later, declining Cdk levels and activation of the Cdk-counteracting phosphatase Cdc14 allow a second APC activator, Cdh1, to associate with the APC (APC^Cdh1). Cyclin proteolysis, a thermodynamically irreversible reaction, is thought to be responsible for the irreversibility of mitotic exit. However, theoretical considerations suggest that de novo protein synthesis should be able to counteract degradation. Protein synthesis constitutes a likewise thermodynamically irreversible process, driven by ATP hydrolysis. In a cellular setting, therefore, protein levels are defined by the rates of two individually irreversible reactions, protein synthesis and degradation.

Irreversibility of mitotic exit is the consequence of systems level feedback

To experimentally investigate the mechanism that makes mitotic exit irreversible, we analysed the contribution of cyclin proteolysis to the irreversibility of budding yeast mitotic exit (Figure 1A). We arrested budding yeast cells in mitosis with high levels of mitotic cyclins by depleting Cdc20 under control of the MET3 promoter. In these cells, we induced Cdh1 expression from the galactose-inducible GALL promoter. We expressed a Cdh1 variant, Cdh1(m11), that activates the APC even in the presence of high Cdk activity due to mutation of 11 Cdk phosphorylation sites. This led to efficient degradation of the major budding yeast mitotic cyclin Clb2, accompanied by dephosphorylation of mitotic Cdk substrates, seen by their change in electrophoretic mobility. Mitotic spindles that were present in the metaphase arrested cells disassembled as Clb2 levels declined, accompanied by outgrowth of pronounced astral microtubules, reminiscent of spindle breakdown at the end of mitosis. This suggests that cyclin destruction efficiently drives mitotic exit.

After 50 minutes, when Clb2 levels became almost undetectable, we turned the APC off again by inactivating a temperature sensitive APC core subunit encoded by the cdc16-123 allele. As a consequence, Clb2 levels recovered, Cdk substrates re-appeared in mitotic hyperphosphorylated forms and mitotic spindles formed again. This suggests that cells had returned to mitosis. FACS analysis of DNA content showed that cells maintained a 2c DNA content and therefore that cells had returned to mitosis without an intervening S-phase. Thus, cyclin destruction promotes mitotic exit events, but is not sufficient to render them irreversible.

Figure 1. Reversible mitotic exit in budding yeast. A: APC^Cdh1-driven Clb2 destruction is reversible and leads to reversible Cdk substrate dephosphorylation and mitotic spindle disassembly and reassemly. Cdh1 expression was induced in metaphase arrested cells for 30 minutes, and APC activity terminated after 50 minutes by inactivating the APC core subunit cdc16-123 at 37°C. A: Levels and electrophoretic mobility of Clb2, Cdh1 and Ase1 were analysed by Western blotting. Tub1 served as a loading control. B:The spindle pole body (SPB) component g-tubulin (Tub4), mitotic spindles (tubulin) and nuclear DNA (stained with DAPI) were visualised. Scale bar, 5µm. C: Wiring diagram of the double negative feedback that provides irreversibility of mitotic exit. D: Computational simulation of double negative feedback.

If not cyclin destruction, what makes mitotic exit irreversible? When we repeated the above experiment, but inactivated the APC after 60 minutes, mitotic exit turned irreversible. Clb2 did not re-accumulate, cells went on to complete cytokinesis and entered G1. Theoretical analysis of the cell cycle control network that operates during mitotic exit suggested that engagement of a double negative feedback loop turns mitotic exit irreversible at this time (Figure 1B). Cdk downregulation causes the accumulation of the stoichiometric Cdk inhibitor Sic1. If Clb2 proteolysis is terminated before Sic1 reaches a threshold, Sic1 accumulation becomes transient and Cdk activity will recover. Clb2 destruction becomes irreversible only if Sic1 levels have reached a threshold that maintains Cdk activity low enough to prevent Clb2 re-synthesis. Our experimental observations confirmed the accumulation and requirement of Sic1 to turn mitotic exit irreversible.

Outlook

During normal mitotic exit, Cdk downregulation is initiated by APC^Cdc20 and APC^Cdh1 activation by the decreasing Cdk/Cdc14 ratio forms an additional double-negative feedback loop that acts redundantly with Sic1. In mammalian cells, the antagonistic relationship between Cdk and Cdh1, and between Cdk and its inhibitory tyrosine phosphorylation, create double-negative feedback loops of Cdk inactivation that likely contribute to irreversibility of mitotic exit. This notion overturns common textbook belief on the unidirectionality of the cell division cycle, with important implications for our understanding how genome instability arises from cell cycle deregulation.

For a list of refereed research papers, see Publications (in navigation on left).