Liebig's law of the minimum in the TGF-ß/SMAD pathway.

Cells use signaling pathways to sense and respond to their environments. The transforming growth factor-ß (TGF-ß) pathway produces context-specific responses. Here, we combined modeling and experimental analysis to study the dependence of the output of the TGF-ß pathway on the abundance of signaling molecules in the pathway. We showed that the TGF-ß pathway processes the variation of TGF-ß receptor abundance using Liebig's law of the minimum, meaning that the output-modifying factor is the signaling protein that is most limited, to determine signaling responses across cell types and in single cells. We found that the abundance of either the type I (TGFBR1) or type II (TGFBR2) TGF-ß receptor determined the responses of cancer cell lines, such that the receptor with relatively low abundance dictates the response. Furthermore, nuclear SMAD2 signaling correlated with the abundance of TGF-ß receptor in single cells depending on the relative expression levels of TGFBR1 and TGFBR2. A similar control principle could govern the heterogeneity of signaling responses in other signaling pathways.

Absolute Quantification of TGF-ß Signaling Proteins Using Quantitative Western Blot.

Cell signaling governs the basic functions of cells by molecular interactions that involve of many proteins. The abundance of signaling proteins can directly influence cellular responses to external signal, contributing to cellular heterogeneity. Absolute quantification of proteins is important for modeling and understanding the complex signaling network. Here, we introduce how to measure the amount of TGF-ß signaling proteins using quantitative immunoblotting. In addition, we discuss how to convert the measurements of protein abundance to the quantities of absolute molecules per cell. This method is generally applicable to the absolute quantification of other proteins.

Cell-type-specific role of CHK2 in mediating DNA damage-induced G2 cell cycle arrest.

Cancer is a life-threatening disease that affects one in three people. Although most cases are sporadic, cancer risk can be increased by genetic factors. It remains unknown why certain genes predispose for specific forms of cancer only, such as checkpoint protein 2 (CHK2), in which gene mutations convey up to twofold higher risk for breast cancer but do not increase lung cancer risk. We have investigated the role of CHK2 and the related kinase checkpoint protein 1 (CHK1) in cell cycle regulation in primary breast and lung primary epithelial cells. At the molecular level, CHK1 activity was higher in lung cells, whereas CHK2 was more active in breast cells. Inhibition of CHK1 profoundly disrupted the cell cycle profile in both lung and breast cells, whereas breast cells were more sensitive toward inhibition of CHK2. Finally, we provide evidence that breast cells require CHK2 to induce a G2-M cell cycle arrest in response of DNA damage, whereas lung cells can partially compensate for the loss of CHK2. Our results provide an explanation as to why CHK2 germline mutations predispose for breast cancer but not for lung cancer.

Tissue-Specific Chk1 Activation Determines Apoptosis by Regulating the Balance of p53 and p21.

The DNA damage response (DDR) protects cells against genomic instability. Surprisingly, little is known about the differences in DDR across tissues, which may affect cancer evolutionary trajectories and chemotherapy response. Using mathematical modeling and quantitative experiments, we found that the DDR is regulated differently in human breast and lung primary cells. Equal levels of cisplatin-DNA lesions caused stronger Chk1 activation in lung cells, leading to resistance. In contrast, breast cells were more resistant and showed more Chk2 activation in response to doxorubicin. Further analyses indicate that Chk1 activity played a regulatory role in p53 phosphorylation, whereas Chk2 activity was essential for p53 activation and p21 expression. We propose a novel "friction model," in which the balance of p53 and p21 levels contributes to the apoptotic response in different tissues. Our results suggest that modulating the balance of p53 and p21 dynamics could optimize the response to chemotherapy.

Spatiotemporal Control of TGF-ß Signaling with Light.

Cells employ signaling pathways to make decisions in response to changes in their immediate environment. Transforming growth factor beta (TGF-ß) is an important growth factor that regulates many cellular functions in development and disease. Although the molecular mechanisms of TGF-ß signaling have been well studied, our understanding of this pathway is limited by the lack of tools that allow the control of TGF-ß signaling with high spatiotemporal resolution. Here, we developed an optogenetic system (optoTGFBRs) that enables the precise control of TGF-ß signaling in time and space. Using the optoTGFBRs system, we show that TGF-ß signaling can be selectively and sequentially activated in single cells through the modulation of the pattern of light stimulations. By simultaneously monitoring the subcellular localization of TGF-ß receptor and Smad2 proteins, we characterized the dynamics of TGF-ß signaling in response to different patterns of blue light stimulations. The spatial and temporal precision of light control will make the optoTGFBRs system as a powerful tool for quantitative analyses of TGF-ß signaling at the single cell level.

Quantitative analysis of transient and sustained transforming growth factor-ß signaling dynamics.

Mammalian cells can decode the concentration of extracellular transforming growth factor-ß (TGF-ß) and transduce this cue into appropriate cell fate decisions. How variable TGF-ß ligand doses quantitatively control intracellular signaling dynamics and how continuous ligand doses are translated into discontinuous cellular fate decisions remain poorly understood. Using a combined experimental and mathematical modeling approach, we discovered that cells respond differently to continuous and pulsating TGF-ß stimulation. The TGF-ß pathway elicits a transient signaling response to a single pulse of TGF-ß stimulation, whereas it is capable of integrating repeated pulses of ligand stimulation at short time interval, resulting in sustained phospho-Smad2 and transcriptional responses. Additionally, the TGF-ß pathway displays different sensitivities to ligand doses at different time scales. While ligand-induced short-term Smad2 phosphorylation is graded, long-term Smad2 phosphorylation is switch-like to a small change in TGF-ß levels. Correspondingly, the short-term Smad7 gene expression is graded, while long-term PAI-1 gene expression is switch-like, as is the long-term growth inhibitory response. Our results suggest that long-term switch-like signaling responses in the TGF-ß pathway might be critical for cell fate determination.