Molecular mechanisms creating bistable switches at cell cycle transitions

Progression through the eukaryotic cell cycle is characterized by specific transitions, where cells move irreversibly from stage i−1 of the cycle into stage i. These irreversible cell cycle transitions are regulated by underlying bistable switches, which share some common features. An inhibitory protein stalls progression, and an activatory protein promotes progression. The inhibitor and activator are locked in a double-negative feedback loop, creating a one-way toggle switch that guarantees an irreversible commitment to move forward through the cell cycle, and it opposes regression from stage i to stage i−1. In many cases, the activator is an enzyme that modifies the inhibitor in multiple steps, whereas the hypo-modified inhibitor binds strongly to the activator and resists its enzymatic activity. These interactions are the basis of a reaction motif that provides a simple and generic account of many characteristic properties of cell cycle transitions. To demonstrate this assertion, we apply the motif in detail to the G1/S transition in budding yeast and to the mitotic checkpoint in mammalian cells. Variations of the motif might support irreversible cellular decision-making in other contexts.     

Effect of positive feedback loops on the robustness of oscillations in the network of cyclin-dependent kinases driving the mammalian cell cycle

The transitions between the G(1) , S, G(2) and M phases of the mammalian cell cycle are driven by a network of cyclin-dependent kinases (Cdks), whose sequential activation is regulated by intertwined negative and positive feedback loops. We previously proposed a detailed computational model for the Cdk network, and showed that this network is capable of temporal self-organization in the form of sustained oscillations, which govern ordered progression through the successive phases of the cell cycle [Gérard and Goldbeter (2009) Proc Natl Acad Sci USA106, 21643-21648]. We subsequently proposed a skeleton model for the cell cycle that retains the core regulatory mechanisms of the detailed model [Gérard and Goldbeter (2011) Interface Focus1, 24-35]. Here we extend this skeleton model by incorporating Cdk regulation through phosphorylation/dephosphorylation and by including the positive feedback loops that underlie the dynamics of the G(1) /S and G(2) /M transitions via phosphatase Cdc25 and via phosphatase Cdc25 and kinase Wee1, respectively. We determine the effects of these positive feedback loops and ultrasensitivity in phosphorylation/dephosphorylation on the dynamics of the Cdk network. The multiplicity of positive feedback loops as well as the existence of ultrasensitivity promote the occurrence of bistability and increase the amplitude of the oscillations in the various cyclin/Cdk complexes. By resorting to stochastic simulations, we further show that the presence of multiple, redundant positive feedback loops in the G(2) /M transition of the cell cycle markedly enhances the robustness of the Cdk oscillations with respect to molecular noise.     

Phosphorylation of CDC25A on SER283 in late S/G2 by CDK/cyclin complexes accelerates mitotic entry

The Cdc25A phosphatase is an essential activator of CDK-cyclin complexes at all steps of the eukaryotic cell cycle. The activity of Cdc25A is itself regulated in part by positive and negative feedback regulatory loops performed by its CDK-cyclin substrates that occur in G1 as well as during the G1/S and G2/M transitions. However, the regulation of Cdc25A during G2 phase progression before mitotic entry has not been intensively characterized. Here, we identify by mass spectrometry analysis a new phosphorylation event of Cdc25A on Serine283. Phospho-specific antibodies revealed that the phosphorylation of this residue appears in late S/G2 phase of an unperturbed cell cycle and is performed by CDK-cyclin complexes. Overexpression studies of wild-type and non-phosphorylatable mutant forms of Cdc25A indicated that Ser283 phosphorylation increases the G2/M-promoting activity of the phosphatase without impacting its stability or subcellular localization. Our results therefore identify a new positive regulatory loop between Cdc25A and its CDK-cyclin substrates which contributes to accelerate entry into mitosis through the regulation of Cdc25A activity in G2.     

GDI-Mediated Cell Polarization in Yeast Provides Precise Spatial and Temporal Control of Cdc42 Signaling

Cell polarization is a prerequisite for essential processes such as cell migration, proliferation or differentiation. The yeast Saccharomyces cerevisiae under control of the GTPase Cdc42 is able to polarize without the help of cytoskeletal structures and spatial cues through a pathway depending on its guanine nucleotide dissociation inhibitor (GDI) Rdi1. To develop a fundamental understanding of yeast polarization we establish a detailed mechanistic model of GDI-mediated polarization. We show that GDI-mediated polarization provides precise spatial and temporal control of Cdc42 signaling and give experimental evidence for our findings. Cell cycle induced changes of Cdc42 regulation enhance positive feedback loops of active Cdc42 production, and thereby allow simultaneous switch-like regulation of focused polarity and Cdc42 activation. This regulation drives the direct formation of a unique polarity cluster with characteristic narrowing dynamics, as opposed to the previously proposed competition between transient clusters. As the key components of the studied system are conserved among eukaryotes, we expect our findings also to apply to cell polarization in other organisms.     

Building a cell cycle oscillator: hysteresis and bistability in the activation of Cdc2

In the early embryonic cell cycle, Cdc2-cyclin B functions like an autonomous oscillator, whose robust biochemical rhythm continues even when DNA replication or mitosis is blocked. At the core of the oscillator is a negative feedback loop; cyclins accumulate and produce active mitotic Cdc2-cyclin B; Cdc2 activates the anaphase-promoting complex (APC); the APC then promotes cyclin degradation and resets Cdc2 to its inactive, interphase state. Cdc2 regulation also involves positive feedback, with active Cdc2-cyclin B stimulating its activator Cdc25 (refs 5-7) and inactivating its inhibitors Wee1 and Myt1 (refs 8-11). Under the correct circumstances, these positive feedback loops could function as a bistable trigger for mitosis, and oscillators with bistable triggers may be particularly relevant to biological applications such as cell cycle regulation. Therefore, we examined whether Cdc2 activation is bistable. We confirm that the response of Cdc2 to non-degradable cyclin B is temporally abrupt and switch-like, as would be expected if Cdc2 activation were bistable. We also show that Cdc2 activation exhibits hysteresis, a property of bistable systems with particular relevance to biochemical oscillators. These findings help establish the basic systems-level logic of the mitotic oscillator.     

Essential roles for the nuclear receptor coactivator Ncoa3 in pluripotency

Septins are guanine nucleotide-binding proteins that form hetero-oligomeric complexes, which assemble into filaments and higher-order structures at sites of cell division and morphogenesis in eukaryotes. Dynamic changes in the organization of septin-containing structures occur concomitantly with progression through the mitotic cell cycle and during cell differentiation. Septins also undergo stage-specific post-translational modifications, which have been implicated in regulating their dynamics, in some cases via purported effects on septin turnover. In our recent study, the fate of two of the five septins expressed in mitotic cells of budding yeast (Saccharomyces cerevisiae) was tracked using two complementary fluorescence-based methods for pulse-chase analysis. During mitotic growth, previously-made molecules of both septins (Cdc10 and Cdc12) persisted through multiple successive divisions and were incorporated equivalently with newly synthesized molecules into hetero-oligomers and higher-order structures. Similarly, in cells undergoing meiosis and the developmental program of sporulation, pre-existing copies of Cdc10 were incorporated into new structures. In marked contrast, Cdc12 was irreversibly excluded from septin complexes and replaced by another septin, Spr3. Here, we discuss the broader implications of these results and related findings with regard to how septin dynamics is coordinated with the mitotic cell cycle and in the yeast life cycle, and how these observations may relate to control of the dynamics of other complex multi-subunit assemblies.     

Activation Domain-dependent Degradation of Somatic Wee1 Kinase

Cell cycle progression is dependent upon coordinate regulation of kinase and proteolytic pathways. Inhibitors of cell cycle transitions are degraded to allow progression into the subsequent cell cycle phase. For example, the tyrosine kinase and Cdk1 inhibitor Wee1 is degraded during G₂ and mitosis to allow mitotic progression. Previous studies suggested that the N terminus of Wee1 directs Wee1 destruction. Using a chemical mutagenesis strategy, we report that multiple regions of Wee1 control its destruction. Most notably, we find that the activation domain of the Wee1 kinase is also required for its degradation. Mutations in this domain inhibit Wee1 degradation in somatic cell extracts and in cells without affecting the overall Wee1 structure or kinase activity. More broadly, these findings suggest that kinase activation domains may be previously unappreciated sites of recognition by the ubiquitin proteasome pathway.