Tongli Zhang

Exploring mechanisms of the DNA-damage response: p53 pulses and their possible relevance to apoptosis.

The transcription factor p53 plays a central role in maintaining genomic integrity. Recent experiments in MCF7 cells have shown that p53 protein level rises and falls in distinct pulses in response to DNA damage. The amplitudes of and intervals between pulses seem to be independent of the extent of damage, and some cells generate regular pulses of p53 over many days. Identifying the molecular mechanisms responsible for such interesting behavior is an important and challenging problem. This paper describes four dual-feedback mechanisms that combine both positive and negative feedback loops, which have been identified in the signaling network responsible for p53 regulation. Mathematical models of all four mechanisms are analyzed to determine if they are consistent with experimental observations and to characterize subtle differences among the possible mechanisms. In addition, a novel molecular mechanism is proposed whereby p53 pulses may induce, at first, cell cycle arrest and, if sustained, cell death. The proposal accounts for basic features of p53-mediated responses to DNA damage and suggests new experiments to probe the dynamics of p53 signaling.

The BEG (PP2A-B55/ENSA/Greatwall) Pathway Ensures Cytokinesis follows Chromosome Separation

Summary Cytokinesis follows separase activation and chromosome segregation. This order is ensured in budding yeast by the mitotic exit network (MEN), where Cdc14p dephosphorylates key conserved Cdk1-substrates exemplified by the anaphase spindle-elongation protein Ase1p. However, in metazoans, MEN and Cdc14 function is not conserved. Instead, the PP2A-B55α/ENSA/Greatwall (BEG) pathway controls the human Ase1p ortholog PRC1. In this pathway, PP2A-B55 inhibition is coupled to Cdk1-cyclin B activity, whereas separase inhibition is maintained by cyclin B concentration. This creates two cyclin B thresholds during mitotic exit. Simulation and experiments using PRC1 as a model substrate show that the first threshold permits separase activation and chromosome segregation, and the second permits PP2A-B55 activation and initiation of cytokinesis. Removal of the ENSA/Greatwall (EG) timer module eliminates this second threshold, as well as associated delay in PRC1 dephosphorylation and initiation of cytokinesis, by uncoupling PP2A-B55 from Cdk1-cyclin B activity. Therefore, temporal order during mitotic exit is promoted by the metazoan BEG pathway.

A Dynamical Framework for the All-or-None G1/S Transition.

Summary The transition from G1 into DNA replication (S phase) is an emergent behavior resulting from dynamic and complex interactions between cyclin-dependent kinases (Cdks), Cdk inhibitors (CKIs), and the anaphase-promoting complex/cyclosome (APC/C). Understanding the cellular decision to commit to S phase requires a quantitative description of these interactions. We apply quantitative imaging of single human cells to track the expression of G1/S regulators and use these data to parametrize a stochastic mathematical model of the G1/S transition. We show that a rapid, proteolytic, double-negative feedback loop between Cdk2:Cyclin and the Cdk inhibitor p27Kip1 drives a switch-like entry into S phase. Furthermore, our model predicts that increasing Emi1 levels throughout S phase are critical in maintaining irreversibility of the G1/S transition, which we validate using Emi1 knockdown and live imaging of G1/S reporters. This work provides insight into the general design principles of the signaling networks governing the temporally abrupt transitions between cell-cycle phases.

The role of APC/C inhibitor Emi2/XErp1 in oscillatory dynamics of early embryonic cell cycles

The early embryonic Xenopus cell cycles are characterized by alternating oscillations of Cyclin-dependent kinase-1 (Cdk1) and Anaphase Promoting Complex/Cyclosome (APC/C) activities. The early cycles before midblastula transition lack significant inhibitory Cdk1 phosphorylations and are driven by periodic accumulation of Cyclin B before M phase and its degradation by APC/C at the end of M phase. Both experiments and mathematical modelling suggest that while Cdk1:CycB phosphorylation activates APC/C, it inhibits its co-activator Cdc20 (Fizzy). These interactions create an amplified negative-feedback loop which is at the heart of all cell cycle oscillations. Recent experiments find that the APC/C inhibitor, Emi2/XErp1 is essential for large amplitude and short period Cyclin B oscillations during early divisions in the intact Xenopus embryo. This finding is counter-intuitive since larger amplitudes should come with slower cycle times. We explain this paradox by analysing the amplified negative feedback model extended with APC/C inhibition by Emi2. We show that Emi2 interferes with the intrinsic time-delay in APC/C activation and inactivation to increase the amplitude as well as shorten the period of Cyclin B oscillation.

Cell cycle commitment in budding yeast emerges from the cooperation of multiple bistable switches

The start-transition (START) in the G1 phase marks the point in the cell cycle at which a yeast cell initiates a new round of cell division. Once made, this decision is irreversible and the cell is committed to progressing through the entire cell cycle, irrespective of arrest signals such as pheromone. How commitment emerges from the underlying molecular interaction network is poorly understood. Here, we perform a dynamical systems analysis of an established cell cycle model, which has never been analysed from a commitment perspective. We show that the irreversibility of the START transition and subsequent commitment can be consistently explained in terms of the interplay of multiple bistable molecular switches. By applying an existing mathematical model to a novel problem and by expanding the model in a self-consistent manner, we achieve several goals: we bring together a large number of experimental findings into a coherent theoretical framework; we increase the scope and the applicability of the original model; we give a systems level explanation of how the START transition and the cell cycle commitment arise from the dynamical features of the underlying molecular interaction network; and we make clear, experimentally testable predictions.

Dynamical scenarios for chromosome bi-orientation.

Chromosome bi-orientation at the metaphase spindle is essential for precise segregation of the genetic material. The process is error-prone, and error-correction mechanisms exist to switch misaligned chromosomes to the correct, bi-oriented configuration. Here, we analyze several possible dynamical scenarios to explore how cells might achieve correct bi-orientation in an efficient and robust manner. We first illustrate that tension-mediated feedback between the sister kinetochores can give rise to a bistable switch, which allows robust distinction between a loose attachment with low tension and a strong attachment with high tension. However, this mechanism has difficulties in explaining how bi-orientation is initiated starting from unattached kinetochores. We propose four possible mechanisms to overcome this problem (exploiting molecular noise; allowing an efficient attachment of kinetochores already in the absence of tension; a trial-and-error oscillation; and a stochastic bistable switch), and assess their impact on the bi-orientation process. Based on our results and supported by experimental data, we put forward a trial-and-error oscillation and a stochastic bistable switch as two elegant mechanisms with the potential to promote bi-orientation both efficiently and robustly.

Robust mitotic entry is ensured by a latching switch.

Summary Cell cycle events are driven by Cyclin dependent kinases (CDKs) and by their counter-acting phosphatases. Activation of the Cdk1:Cyclin B complex during mitotic entry is controlled by the Wee1/Myt1 inhibitory kinases and by Cdc25 activatory phosphatase, which are themselves regulated by Cdk1:Cyclin B within two positive circuits. Impairing these two feedbacks with chemical inhibitors induces a transient entry into M phase referred to as mitotic collapse. The pathology of mitotic collapse reveals that the positive circuits play a significant role in maintaining the M phase state. To better understand the function of these feedback loops during G2/M transition, we propose a simple model for mitotic entry in mammalian cells including spatial control over Greatwall kinase phosphorylation. After parameter calibration, the model is able to recapture the complex and non-intuitive molecular dynamics reported by Potapova et al. (Potapova et al., 2011). Moreover, it predicts the temporal patterns of other mitotic regulators which have not yet been experimentally tested and suggests a general design principle of cell cycle control: latching switches buffer the cellular stresses which accompany cell cycle processes to ensure that the transitions are smooth and robust.

Role for regulated phosphatase activity in generating mitotic oscillations in Xenopus cell-free extracts

Significance A long-standing puzzle in the cell-cycle field is how it can be that the anaphase-promoting complex/cyclosome (APC/C) gets activated only when cyclin-dependent kinase 1 (Cdk1) activity reaches a very high level in metaphase but stays active even after Cdk1 activity drops to much lower levels as cells exit mitosis. In this manuscript, we propose that the regulation of a protein phosphatase 2A (PP2A:B55δ) by Greatwall kinase might provide a solution to this puzzle. Our proposal is consistent with what is already known about the regulation of Cdk1 and PP2A:B55δ in frog eggs, and it leads to testable predictions. Although current textbook explanations of cell-cycle control in eukaryotes emphasize the periodic activation of cyclin-dependent protein kinases (CDKs), recent experimental observations suggest a significant role for the periodic activation and inactivation of a CDK-counteracting protein phosphatase 2A with a B55δ subunit (PP2A:B55δ), during mitotic cycles in frog-egg extracts and early embryos. In this paper, we extend an earlier mathematical model of embryonic cell cycles to include experimentally motivated roles for PP2A:B55δ and its regulation by Greatwall kinase. Our model is consistent with what is already known about the regulation of CDK and PP2A:B55δ in frog eggs, and it suggests a previously undescribed role for the Greatwall-PP2A:B55δ interaction in creating a toggle switch for activation of the anaphase-promoting complex as embryonic cells exit mitosis and return to interphase.