Single-cell variation leads to population invariance in NF-?B signaling dynamics.
Molecular biology of the cell
2015; 26 (3): 583-590
High-sensitivity measurements of multiple kinase activities in live single cells.
2014; 157 (7): 1724-1734
The activation dynamics of nuclear factor (NF)-?B have been shown to affect downstream gene expression. On activation, NF-?B shuttles back and forth across the nuclear envelope. Many dynamic features of this shuttling have been characterized, and most features vary significantly with respect to ligand type and concentration. Here, we report an invariant feature with regard to NF-?B dynamics in cellular populations: the distribution-the average, as well as the variance-of the time between two nuclear entries (the period). We find that this period is conserved, regardless of concentration and across several different ligands. Intriguingly, the distributions observed at the population level are not observed in individual cells over 20-h time courses. Instead, the average period of NF-?B nuclear translocation varies considerably among single cells, and the variance is much smaller within a cell than that of the population. Finally, analysis of daughter-cell pairs and isogenic populations indicates that the dynamics of the NF-?B response is heritable but diverges over multiple divisions, on the time scale of weeks to months. These observations are contrary to the existing theory of NF-?B dynamics and suggest an additional level of control that regulates the overall distribution of translocation timing at the population level.
View details for DOI 10.1091/mbc.E14-08-1267
View details for PubMedID 25473117
The microfluidic multitrap nanophysiometer for hematologic cancer cell characterization reveals temporal sensitivity of the calcein-AM efflux assay
Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-?B) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.
View details for DOI 10.1016/j.cell.2014.04.039
View details for PubMedID 24949979
Single-Cell and Population NF-kappa B Dynamic Responses Depend on Lipopolysaccharide Preparation
2013; 8 (1)
Single-cell NF-kappa B dynamics reveal digital activation and analogue information processing
2010; 466 (7303): 267-U149
Cytometric studies utilizing flow cytometry or multi-well culture plate fluorometry are often limited by a deficit in temporal resolution and a lack of single cell consideration. Unfortunately, many cellular processes, including signaling, motility, and molecular transport, occur transiently over relatively short periods of time and at different magnitudes between cells. Here we demonstrate the multitrap nanophysiometer (MTNP), a low-volume microfluidic platform housing an array of cell traps, as an effective tool that can be used to study individual unattached cells over time with precise control over the intercellular microenvironment. We show how the MTNP platform can be used for hematologic cancer cell characterization by measuring single T cell levels of CRAC channel modulation, non-translational motility, and ABC-transporter inhibition via a calcein-AM efflux assay. The transporter data indicate that Jurkat T cells exposed to indomethacin continue to accumulate fluorescent calcein for over 60 minutes after calcein-AM is removed from the extracellular space.
View details for DOI 10.1038/srep05117
View details for Web of Science ID 000336529000003
View details for PubMedID 24873950
Computational modeling of mammalian signaling networks
WILEY INTERDISCIPLINARY REVIEWS-SYSTEMS BIOLOGY AND MEDICINE
2010; 2 (2): 194-209
Cells operate in dynamic environments using extraordinary communication capabilities that emerge from the interactions of genetic circuitry. The mammalian immune response is a striking example of the coordination of different cell types. Cell-to-cell communication is primarily mediated by signalling molecules that form spatiotemporal concentration gradients, requiring cells to respond to a wide range of signal intensities. Here we use high-throughput microfluidic cell culture and fluorescence microscopy, quantitative gene expression analysis and mathematical modelling to investigate how single mammalian cells respond to different concentrations of the signalling molecule tumour-necrosis factor (TNF)-alpha, and relay information to the gene expression programs by means of the transcription factor nuclear factor (NF)-kappaB. We measured NF-kappaB activity in thousands of live cells under TNF-alpha doses covering four orders of magnitude. We find, in contrast to population-level studies with bulk assays, that the activation is heterogeneous and is a digital process at the single-cell level with fewer cells responding at lower doses. Cells also encode a subtle set of analogue parameters to modulate the outcome; these parameters include NF-kappaB peak intensity, response time and number of oscillations. We developed a stochastic mathematical model that reproduces both the digital and analogue dynamics as well as most gene expression profiles at all measured conditions, constituting a broadly applicable model for TNF-alpha-induced NF-kappaB signalling in various types of cells. These results highlight the value of high-throughput quantitative measurements with single-cell resolution in understanding how biological systems operate.
View details for DOI 10.1038/nature09145
View details for Web of Science ID 000279580800043
View details for PubMedID 20581820
A Noisy Paracrine Signal Determines the Cellular NF-kappa B Response to Lipopolysaccharide
2009; 2 (93)
One of the most exciting developments in signal transduction research has been the proliferation of studies in which a biological discovery was initiated by computational modeling. In this study, we review the major efforts that enable such studies. First, we describe the experimental technologies that are generally used to identify the molecular components and interactions in, and dynamic behavior exhibited by, a network of interest. Next, we review the mathematical approaches that are used to model signaling network behavior. Finally, we focus on three specific instances of 'model-driven discovery': cases in which computational modeling of a signaling network has led to new insights that have been verified experimentally.
View details for DOI 10.1002/wsbm.52
View details for Web of Science ID 000283711700007
View details for PubMedID 20836022
Microfluidic platform for real-time signaling analysis of multiple single T cells in parallel
LAB ON A CHIP
2008; 8 (10): 1700-1712
Nearly identical cells can exhibit substantially different responses to the same stimulus. We monitored the nuclear localization dynamics of nuclear factor kappaB (NF-kappaB) in single cells stimulated with tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharide (LPS). Cells stimulated with TNF-alpha have quantitative differences in NF-kappaB nuclear localization, whereas LPS-stimulated cells can be clustered into transient or persistent responders, representing two qualitatively different groups based on the NF-kappaB response. These distinct behaviors can be linked to a secondary paracrine signal secreted at low concentrations, such that not all cells undergo a second round of NF-kappaB activation. From our single-cell data, we built a computational model that captures cell variability, as well as population behaviors. Our findings show that mammalian cells can create "noisy" environments to produce diversified responses to stimuli.
View details for DOI 10.1126/scisignal.2000599
View details for Web of Science ID 000275604000003
View details for PubMedID 19843957
Intra-microfluidic pinocytic loading of human T cells
2007 IEEE/NIH LIFE SCIENCE SYSTEMS AND APPLICATIONS WORKSHOP
Deciphering the signaling pathways that govern stimulation of na´ve CD4+ T helper cells by antigen-presenting cells via formation of the immunological synapse is key to a fundamental understanding of the progression of successful adaptive immune response. The study of T cell-APC interactions in vitro is challenging, however, due to the difficulty of tracking individual, non-adherent cell pairs over time. Studying single cell dynamics over time reveals rare, but critical, signaling events that might be averaged out in bulk experiments, but these less common events are undoubtedly important for an integrated understanding of a cellular response to its microenvironment. We describe a novel application of microfluidic technology that overcomes many limitations of conventional cell culture and enables the study of hundreds of passively sequestered hematopoietic cells for extended periods of time. This microfluidic cell trap device consists of 440 18 micromx18 micromx10 microm PDMS, bucket-like structures opposing the direction of flow which serve as corrals for cells as they pass through the cell trap region. Cell viability analysis revealed that more than 70% of na´ve CD4+ T cells (TN), held in place using only hydrodynamic forces, subsequently remain viable for 24 hours. Cytosolic calcium transients were successfully induced in TN cells following introduction of chemical, antibody, or cellular forms of stimulation. Statistical analysis of TN cells from a single stimulation experiment reveals the power of this platform to distinguish different calcium response patterns, an ability that might be utilized to characterize T cell signaling states in a given population. Finally, we investigate in real time contact- and non-contact-based interactions between primary T cells and dendritic cells, two main participants in the formation of the immunological synapse. Utilizing the microfluidic traps in a daisy-chain configuration allowed us to observe calcium transients in TN cells exposed only to media conditioned by secretions of lipopolysaccharide-matured dendritic cells, an event which is easily missed in conventional cell culture where large media-to-cell ratios dilute cellular products. Further investigation into this intercellular signaling event indicated that LPS-matured dendritic cells, in the absence of antigenic stimulation, secrete chemical signals that induce calcium transients in T(N) cells. While the stimulating factor(s) produced by the mature dendritic cells remains to be identified, this report illustrates the utility of these microfluidic cell traps for analyzing arrays of individual suspension cells over time and probing both contact-based and intercellular signaling events between one or more cell populations.
View details for DOI 10.1039/b719799c
View details for Web of Science ID 000260466300015
View details for PubMedID 18813394