Galit Lahav

Oscillations and variability in the p53 system

Understanding the dynamics and variability of protein circuitry requires accurate measurements in living cells as well as theoretical models. To address this, we employed one of the best‐studied protein circuits in human cells, the negative feedback loop between the tumor suppressor p53 and the oncogene Mdm2. We measured the dynamics of fluorescently tagged p53 and Mdm2 over several days in individual living cells. We found that isogenic cells in the same environment behaved in highly variable ways following DNA‐damaging gamma irradiation: some cells showed undamped oscillations for at least 3 days (more than 10 peaks). The amplitude of the oscillations was much more variable than the period. Sister cells continued to oscillate in a correlated way after cell division, but lost correlation after about 11 h on average. Other cells showed low‐frequency fluctuations that did not resemble oscillations. We also analyzed different families of mathematical models of the system, including a novel checkpoint mechanism. The models point to the possible source of the variability in the oscillations: low‐frequency noise in protein production rates, rather than noise in other parameters such as degradation rates. This study provides a view of the extensive variability of the behavior of a protein circuit in living human cells, both from cell to cell and in the same cell over time.

p53 dynamics control cell fate

Dynamic Responses Expression of the tumor suppressor p53 is activated in response to cell stress. The dynamics of p53 activation can vary, depending on the stressor, resulting in either pulsatile or constant p53 levels; however, the functional consequence of these different dynamics is unclear. Purvis et al. (p. 1440) developed a method to control p53 dynamics in human cells. Pulsing p53 selectively activated genes involved in cell cycle arrest and DNA repair, allowing recovery from DNA damage. In contrast, sustained p53 promoted induction of terminal genes leading to cellular senescence. Thus, protein dynamics can affect cell fate decisions. After DNA damage, pulses of p53 allow recovery, whereas sustained levels lead to senescence. Cells transmit information through molecular signals that often show complex dynamical patterns. The dynamic behavior of the tumor suppressor p53 varies depending on the stimulus; in response to double-strand DNA breaks, it shows a series of repeated pulses. Using a computational model, we identified a sequence of precisely timed drug additions that alter p53 pulses to instead produce a sustained p53 response. This leads to the expression of a different set of downstream genes and also alters cell fate: Cells that experience p53 pulses recover from DNA damage, whereas cells exposed to sustained p53 signaling frequently undergo senescence. Our results show that protein dynamics can be an important part of a signal, directly influencing cellular fate decisions.

Encoding and decoding cellular information through signaling dynamics.

A growing number of studies are revealing that cells can send and receive information by controlling the temporal behavior (dynamics) of their signaling molecules. In this Review, we discuss what is known about the dynamics of various signaling networks and their role in controlling cellular responses. We identify general principles that are emerging in the field, focusing specifically on how the identity and quantity of a stimulus is encoded in temporal patterns, how signaling dynamics influence cellular outcomes, and how specific dynamical patterns are both shaped and interpreted by the structure of molecular networks. We conclude by discussing potential functional roles for transmitting cellular information through the dynamics of signaling molecules and possible applications for the treatment of disease.

Recurrent Initiation: A Mechanism for Triggering p53 Pulses in Response to DNA Damage

DNA damage initiates a series of p53 pulses. Although much is known about the interactions surrounding p53, little is known about which interactions contribute to p53s dynamical behavior. The simplest explanation is that these pulses are oscillations intrinsic to the p53/Mdm2 negative feedback loop. Here we present evidence that this simple mechanism is insufficient to explain p53 pulses; we show that p53 pulses are externally driven by pulses in the upstream signaling kinases, ATM and Chk2, and that the negative feedback between p53 and ATM, via Wip1, is essential for maintaining the uniform shape of p53 pulses. We propose that p53 pulses result from repeated initiation by ATM, which is reactivated by persistent DNA damage. Our study emphasizes the importance of collecting quantitative dynamic information at high temporal resolution for understanding the regulation of signaling pathways and opens new ways to manipulate p53 pulses to ask questions about their function in response to DNA damage.

Cell Growth and Size Homeostasis in Proliferating Animal Cells

Size Matters Cells of different types come in different sizes. Size is established by a trade-off of growth and division; as a result, the distribution of cell size in a population is held stable over time. A classic question in cell physiology is whether the growth rate of cells is constant over time or whether, as cells grow bigger during the cell cycle, they grow faster and faster. Using experimental and mathematical approaches, Tzur et al. (p. 167; see the Perspective by Edgar and Kim) show that the growth rate of mouse lymphoblastoid cells in culture is slow in the G1 phase and then increases to reach a constant exponential rate. Thus, there is an active size-control mechanism that limits size variation in animal cells. Lymphoblasts grow slowly after mitosis, then reach a constant exponential rate, indicating an active size-control mechanism. A long-standing question in biology is whether there is an intrinsic mechanism for coordinating growth and the cell cycle in metazoan cells. We examined cell size distributions in populations of lymphoblasts and applied a mathematical analysis to calculate how growth rates vary with both cell size and the cell cycle. Our results show that growth rate is size-dependent throughout the cell cycle. After initial growth suppression, there is a rapid increase in growth rate during the G1 phase, followed by a period of constant exponential growth. The probability of cell division varies independently with cell size and cell age. We conclude that proliferating mammalian cells have an intrinsic mechanism that maintains cell size.

The ups and downs of p53: understanding protein dynamics in single cells

Cells living in a complex environment must constantly detect, process and appropriately respond to changing signals. Therefore, all cellular information processing is dynamic in nature. As a consequence, understanding the process of signal transduction often requires detailed quantitative analysis of dynamic behaviours. Here, we focus on the oscillatory dynamics of the tumour suppressor protein p53 as a model for studying protein dynamics in single cells to better understand its regulation and function.

Stimulus-dependent dynamics of p53 in single cells.

Many biological networks respond to various inputs through a common signaling molecule that triggers distinct cellular outcomes. One potential mechanism for achieving specific input–output relationships is to trigger distinct dynamical patterns in response to different stimuli. Here we focused on the dynamics of p53, a tumor suppressor activated in response to cellular stress. We quantified the dynamics of p53 in individual cells in response to UV and observed a single pulse that increases in amplitude and duration in proportion to the UV dose. This graded response contrasts with the previously described series of fixed pulses in response to γ‐radiation. We further found that while γ‐triggered p53 pulses are excitable, the p53 response to UV is not excitable and depends on continuous signaling from the input‐sensing kinases. Using mathematical modeling and experiments, we identified feedback loops that contribute to specific features of the stimulus‐dependent dynamics of p53, including excitability and input‐duration dependency. Our study shows that different stresses elicit different temporal profiles of p53, suggesting that modulation of p53 dynamics might be used to achieve specificity in this network.

Basal Dynamics of p53 Reveal Transcriptionally Attenuated Pulses in Cycling Cells

The tumor suppressor p53 is activated by stress and leads to cellular outcomes such as apoptosis and cell-cycle arrest. Its activation must be highly sensitive to ensure that cells react appropriately to damage. However, proliferating cells often encounter transient damage during normal growth, where cell-cycle arrest or apoptosis may be unfavorable. How does the p53 pathway achieve the right balance between high sensitivity and tolerance to intrinsic damage? Using quantitative time-lapse microscopy of individual human cells, we found that proliferating cells show spontaneous pulses of p53, which are triggered by an excitable mechanism during cell-cycle phases associated with intrinsic DNA damage. However, in the absence of sustained damage, posttranslational modifications keep p53 inactive, preventing it from inducing p21 expression and cell-cycle arrest. Our approach of quantifying basal dynamics in individual cells can now be used to study how other pathways in human cells achieve sensitivity in noisy environments.

Quantitative Live Cell Imaging Reveals a Gradual Shift between DNA Repair Mechanisms and a Maximal Use of HR in Mid S Phase

DNA double-strand breaks are repaired by two main pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). The choice between these pathways depends on cell-cycle phase; however the continuous effect of cell cycle on the balance between them is still unclear. We used live cell imaging and fluorescent reporters for 53BP1, Rad52, and cell cycle to quantify the relative contribution of NHEJ and HR at different points of the cell cycle in single cells. We found that NHEJ is the dominant repair pathway in G1 and G2 even when both repair pathways are functional. The shift from NHEJ to HR is gradual, with the highest proportion of breaks repaired by HR in mid S, where the amount of DNA replication is highest. Higher proportions of HR also strongly correlate with slower rates of repair. Our study shows that the choice of repair mechanism is continuously adjusted throughout the cell cycle and suggests that the extent of active replication, rather than the presence of a sister chromatid influences the balance between the two repair pathways in human cells.

Phosphorylation by Casein Kinase I promotes the turnover of the Mdm2 oncoprotein via the SCFβ-TRCP ubiquitin ligase

Mdm2 is the major negative regulator of the p53 pathway. Here, we report that Mdm2 is rapidly degraded after DNA damage and that phosphorylation of Mdm2 by casein kinase I (CKI) at multiple sites triggers its interaction with, and subsequent ubiquitination and destruction, by SCF(beta-TRCP). Inactivation of either beta-TRCP or CKI results in accumulation of Mdm2 and decreased p53 activity, and resistance to apoptosis induced by DNA damaging agents. Moreover, SCF(beta-TRCP)-dependent Mdm2 turnover also contributes to the control of repeated p53 pulses in response to persistent DNA damage. Our results provide insight into the signaling pathways controlling Mdm2 destruction and further suggest that compromised regulation of Mdm2 results in attenuated p53 activity, thereby facilitating tumor progression.