Mitotic exit

Many speculations were made with regard to the control mechanisms employed by a cell to promote the irreversibility of mitotic exit in a eukaryotic model organism, the budding yeast Saccharomyces cerevisiae. Proteolytic degradation of cell cycle regulators and corresponding effects on the levels of cyclin-dependent kinases were proposed as a mechanism that promotes eukaryotic cell cycle and metaphase-to-anaphase transition in particular. In this theory, anaphase promoting complex (APC), a class of ubiquitin ligase, facilitates degradation of mitotic cyclins (Clb2) and anaphase-inhibiting factors (PDS1, CUT2) to promote mitotic exit. APC ubiquitinates nine-amino acid motif known as the destruction box (D box) in the NH2-terminal domain of mitotic cyclins for degradation by proteasome. At later phase, downregulation of Cdk1 and activation of Cdc14, a Cdh1-activating phosphatase, promotes formation of APC in association with Cdh1 (APC-Cdh1) to degrade Clb2s. Without Cdk1-Clb2 complexes to phosphorylate proteins that are involved in spindle dynamics such as Sli15, Ase1, and Ask1, spindle elongation and chromosomal segregation are promoted, facilitating mitotic exit. The importance of proteolytic degradation in eukaryotic cell cycle changed the view of cell division as a simple kinase cascade to a more complex process in which interactions among phosphorylation, ubiquitination, and proteolysis are necessary. However, experiments using budding yeast cells with cdc28-as1, an INM-PP1 (ATP analog)-sensitive Cdk allele, proved that destruction of B-type cyclins (Clb) is not necessary for triggering irreversible mitotic exit. Clb2 degradation did shorten the Cdk1-inhibition period required for triggering irreversible mitotic exit indicating that cyclin proteolysis contributes to the dynamic nature of the eukaryotic cell cycle due to slower timescale of its action but is unlikely to be the major determining factor in triggering irreversible cell cycle transitions. ==Sic1 levels==

Discoveries were made which indicated the importance of the level of the inhibitors of cyclin-dependent kinases in regulating eukaryotic cell cycle. In particular, the level of Sic1, a stoichiometric inhibitor of Clb-CDK complexes in budding yeast, was shown to be particularly important in irreversible G1-S transition by irreversibly activating S phase kinases. Sic1 level was shown to play a major role in triggering irreversible mitotic exit (M-G1 transition) as well as in G1-S transition. During mitosis, decreasing levels of Cdk1 leads to the activation of Cdc14, a phosphatase that counteracts Cdk1 via activation of Cdh1 and Swi5, a transcriptional activator of Sic1 proteins. While degradation of Sic1 to a certain low level triggered the onset of S phase, accumulation of Sic1 to a certain high level was required to trigger irreversible mitotic exit. Despite different thresholds of Sic1 level that are required to trigger mitotic exit compared to G1-S transition, the level of Sic1 was shown to play a key role in regulating eukaryotic cell cycle by inhibiting the activity of CDKs. ==Dynamical systems approach==

Because eukaryotic cell cycle involves a variety of proteins and regulatory interactions, dynamical systems approach can be taken to simplify a complex biological circuit into a general framework for better analysis. Among the four possible input/output relationships, the relationship between Sic1 level and mitotic exit seems to show the characteristics of an irreversible bistable switch, driven by feedback between APC-Cdh1, Sic1, and Clb2-Cdk1. Bistable input/output relationship is characterized by two stable states with two bifurcation points. Multiple outputs are possible for one specific input in the region of bistability, marked by two bifurcation points. In addition, the bistable relationship displays hysteresis: the final state/output depends on the history of the input as well as the current value of input because the system has a memory. Thus, the only one-dimensional bifurcation that can exist in imperfect biological world is the saddle-node bifurcation. The bistable relation between M-G1 transition and Sic1 level can be represented as a diagram of two saddle-node bifurcations in which the system's behavior changes qualitatively with a small change in control parameter, the amount of Sic1. ==Systems-level feedback==

Because the behavior of cell cycle critically depends on the amount of Sic1 at the M-G1 transition state, the amount of Sic1 is tightly regulated by systems-level feedbacks. Because Cdk1-Clb2 inhibits Sic1 by phosphorylating Sic1 and making Sic1 available for degradation via ubiquitylation, APC-Cdh1-dependent degradation of Cdk1-Clb2 not only decreases the level of available Cdk1-Clb2 complexes but also increases the level of Sic1 which in turn further inhibits the function of Cdk1-Clb2. FEAR (Cdc14 early anaphase release) pathway facilitates Clb2-Cdk1-dependent phosphorylation of Net1 which transiently releases Cdc14 from Net1. The released Cdc14 and Clb2-Cdk1 complexes go onto form spindles that activates mitotic exit network (MEN). MEN allows sustained release of Cdc14 from the nucleolus, Sic1 positively regulates itself by inhibiting Cdk1-Clb2 to release inhibition of Swi5, and Cdh1 also positively regulates itself by inhibiting Clb2-Cdk1 to release inhibition of MEN which can activate Cdc14 and subsequently Cdh1 itself. The double-negative feedback loop, formed by APC-Cdh1 and Sic1, is required to maintain low Clb2-Cdk1 activity because Clb2 auto-activates its synthesis by activating transcriptional factors, Fkh2–Mcm1 Ndd1 complex. ==Implications==

Eukaryotic cell cycle consists of various checkpoints and feedback loops to ensure faithful and successful cell division. During mitosis for example, when duplicated chromosomes are improperly attached to mitotic spindle, spindle assembly checkpoint (SAC) proteins including Mad and Bub inhibit APC-Cdc20 to delay entry into anaphase and B-type cyclin degradations. In addition, when mitotic spindles are misaligned, MEN and subsequently Cdc14 are inhibited in a Bub2 and Bfa1-dependent manner to prevent degradation of mitotic cyclins and anaphase entry. ==Reversibility of Mitotic Exit==

The regulation of mitotic progression and exit is of paramount importance, as errors in this process can lead to aneuploidy and cancer. Recent studies, notably involving the Cdk1 inhibitor flavopiridol, have shed light on the fascinating aspect of mitotic exit reversibility, challenging long-held views on the unidirectionality of cell cycle transitions. Central to understanding mitotic exit is Cdk1, a kinase that orchestrates mitotic progression. Activated by binding to cyclins, particularly cyclin A and B, Cdk1's activity peaks during mitosis. The timely degradation of these cyclins, especially cyclin B, is crucial for mitotic exit. This degradation ensures the dephosphorylation of Cdk1 substrates, acting as a timing mechanism for the critical transition from metaphase to anaphase, and eventually to the exit from mitosis. Such meticulous regulation highlights the cell's commitment to the irreversibility of the cell cycle phases under normal conditions. The introduction of flavopiridol, a potent Cdk1 inhibitor, has revolutionized the understanding of mitotic regulation. When applied to vertebrate cells during mitosis, flavopiridol induced a premature exit from mitosis, accompanied by cytokinesis, but notably without prior chromatid separation. This phenomenon led to an intriguing 'cut' phenotype where chromosomes, although decondensed and enveloped in newly formed nuclear membranes, remained trapped within the cell's midbody (Figure 4). The most striking finding from the flavopiridol studies is the reversibility of mitotic exit. When the proteasome activity, responsible for cyclin degradation, is inhibited, flavopiridol's effects become reversible. Upon the removal of flavopiridol, cells previously induced to exit mitosis prematurely could revert to a metaphase state. This reversal involves the reassembly of the mitotic spindle, retraction of the cytokinetic furrow, dissolution of the newly formed nuclear envelope, and recondensation of chromosomes.The reversibility of mitotic exit heavily relies on the stability of cyclin B. In conditions where cyclin B is shielded from degradation, such as in the presence of proteasome inhibitors like MG132, the mitotic exit induced by flavopiridol can be reversed. This finding was further corroborated using non-degradable cyclin B1, which showed that cells with stabilized cyclin B could revert to metaphase upon the removal of flavopiridol, indicating a direct correlation between cyclin B stability and mitotic exit reversibility. ==Mitotic Exit as a Cancer Therapeutic Target==

Cancer research has long focused on the disruption of cell division as a therapeutic strategy. Antimitotic drugs, targeting microtubule dynamics, have been a cornerstone of this approach. However, their success is often hampered by side effects like neurotoxicity and limited efficacy against solid tumors. The use of taxanes and vinca alkaloids, targeting microtubule dynamics, has been a standard in cancer therapy. However, their efficacy is often undermined by severe side effects, notably neurotoxicity, due to the perturbation of microtubules in neurons. Additionally, spindle-specific antimitotic drugs, while less neurotoxic, have shown limited success against solid tumors, often comparable or inferior to traditional drugs. This scenario underscores the need for alternative antimitotic strategies that are both more effective and less toxic. Recent studies have shifted focus to the process of mitotic exit, particularly blocking this phase as a novel anticancer strategy. The key player in this context is Cdc20, a regulatory protein that activates the Anaphase Promoting Complex/Cyclosome (APC/C), leading to cyclin B1 degradation and mitotic exit. Knockdown of Cdc20 using RNAi techniques induces a prolonged mitotic arrest, leading to cell death. This approach has been shown to be more effective than spindle-perturbing drugs in killing cancer cells, including those resistant to apoptosis or prone to slippage (escape from drug-induced arrest). Cdc20 knockdown leads to sustained mitotic arrest by stabilizing cyclin B1 levels, thereby preventing slippage. This prolonged arrest gives more time for apoptotic mechanisms to activate and kill the cancer cells. Unlike spindle-perturbing drugs, this strategy does not rely on spindle assembly checkpoint (SAC) activation and is effective even in SAC-deficient cells. Moreover, Cdc20 knockdown induces cell death through both mitochondrial outer membrane permeabilization (MOMP)-dependent and -independent pathways, making it effective against a broader range of cancer cell types (Figure 5). The blockade of mitotic exit presents several advantages over traditional spindle-targeting drugs. Firstly, it avoids the neurotoxicity associated with microtubule perturbation. Secondly, its efficacy is not limited by SAC activity or the cell's ability to evade death through slippage. Finally, it targets a fundamental and late stage of cell division, potentially reducing the chance of resistance development compared to drugs targeting earlier stages like spindle assembly. While promising, targeting mitotic exit as a cancer therapy is not without challenges. Developing specific, potent inhibitors of Cdc20 or other mitotic exit proteins is a significant hurdle. Additionally, understanding the exact mechanisms by which prolonged mitotic arrest induces cell death, and identifying potential drug targets within these pathways, remains a key area of research. ==Cell Polarity Proteins in Mitotic Exit==

In budding yeast, the process of mitotic exit involves a network of proteins that intertwine the concepts of cell polarity and mitotic regulation. Central to this network are the roles of Cdc14, a cell cycle phosphatase, and various cell polarity proteins, which collectively ensure the orderly transition from mitosis to the G1 phase of the cell cycle. Cdc14 plays a pivotal role in mitotic exit by dephosphorylating key targets such as Cdh1/Hct1 and Sic1. This action leads to a decrease in mitotic cyclin-dependent kinase (Cdk-Clb) activity, a prerequisite for mitotic exit. The regulation of Cdc14 is tightly controlled; it is sequestered in the nucleolus during interphase and metaphase and is released at the onset of anaphase. This release is crucial for Cdc14 to perform its function in triggering mitotic exit. The release of Cdc14 from the nucleolus involves two essential steps: an initial partial release facilitated by separase (Esp1) and polo kinase (Cdc5) and a subsequent full release governed by the mitotic exit network (MEN). The MEN is a GTPase-driven signaling network crucial for mitotic exit, and its proper functioning is essential for the cell cycle's fidelity. A key player in the MEN is Tem1, a small Ras-like GTPase. The regulation of Tem1 is complex, involving its inactivation by the Bfa1-Bub2 GAP complex and activation by the GEF Lte1. The spatial dynamics of these interactions are crucial; while Tem1 is associated with the spindle pole body (SPB), Lte1 is associated with the bud cortex but not the mother cell. This arrangement ensures that MEN activation and, consequently, mitotic exit occur only when the nucleus is correctly positioned within the bud. Cdc24, Cdc42, and Ste20 proteins, through their interaction with Lte1 and other MEN components, play a vital role in ensuring that mitotic exit is tightly regulated and occurs only under appropriate conditions (Figure 6). For instance, the cell polarity protein Cdc42 regulates Ste20 activity, highlighting the interconnectedness of these pathways. == References ==