Partner: Claire Harper

University of Manchester (GB)

Recent publications
1.Adamson A., Boddington C., Downton P., Rowe W., Bagnall J., Lam C., Maya-Mendoza A., Schmidt L., Harper Claire V.V., Spiller David G., Rand David A.A., Jackson Dean A., White Michael R. H.R., Paszek P., Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states, Nature Communications, ISSN: 2041-1723, DOI: 10.1038/ncomms12057, Vol.7, pp.12057-1-14, 2016
Abstract:

Cells respond dynamically to pulsatile cytokine stimulation. Here we report that single, or well-spaced pulses of TNFα (>100 min apart) give a high probability of NF-κB activation. However, fewer cells respond to shorter pulse intervals (<100 min) suggesting a heterogeneous refractory state. This refractory state is established in the signal transduction network downstream of TNFR and upstream of IKK, and depends on the level of the NF-κB system negative feedback protein A20. If a second pulse within the refractory phase is IL-1β instead of TNFα, all of the cells respond. This suggests a mechanism by which two cytokines can synergistically activate an inflammatory response. Gene expression analyses show strong correlation between the cellular dynamic response and NF-κB-dependent target gene activation. These data suggest that refractory states in the NF-κB system constitute an inherent design motif of the inflammatory response and we suggest that this may avoid harmful homogenous cellular activation.

Affiliations:
Adamson A.-other affiliation
Boddington C.-other affiliation
Downton P.-other affiliation
Rowe W.-other affiliation
Bagnall J.-other affiliation
Lam C.-other affiliation
Maya-Mendoza A.-other affiliation
Schmidt L.-other affiliation
Harper Claire V.V.-University of Manchester (GB)
Spiller David G.-other affiliation
Rand David A.A.-University of Warwick (GB)
Jackson Dean A.-other affiliation
White Michael R. H.R.-University of Manchester (GB)
Paszek P.-other affiliation
2.Ankers John M., Awais R., Jones Nicholas A., Boyd J., Ryan S., Adamson Antony D., Harper Claire V.V., Bridge L., Spiller David G., Jackson Dean A., Paszek P., Sée V., White Michael R.R., Dynamic NF-κB and E2F interactions control the priority and timing of inflammatory signalling and cell proliferation, eLife, ISSN: 2050-084X, DOI: 10.7554/eLife.10473, Vol.5, pp.e10473-1-35, 2016
Abstract:

Dynamic cellular systems reprogram gene expression to ensure appropriate cellular fate responses to specific extracellular cues. Here we demonstrate that the dynamics of Nuclear Factor kappa B (NF-κB) signalling and the cell cycle are prioritised differently depending on the timing of an inflammatory signal. Using iterative experimental and computational analyses, we show physical and functional interactions between NF-κB and the E2 Factor 1 (E2F-1) and E2 Factor 4 (E2F-4) cell cycle regulators. These interactions modulate the NF-κB response. In S-phase, the NF-κB response was delayed or repressed, while cell cycle progression was unimpeded. By contrast, activation of NF-κB at the G1/S boundary resulted in a longer cell cycle and more synchronous initial NF-κB responses between cells. These data identify new mechanisms by which the cellular response to stress is differentially controlled at different stages of the cell cycle.

Affiliations:
Ankers John M.-other affiliation
Awais R.-other affiliation
Jones Nicholas A.-Massachusetts Institute of Technology (US)
Boyd J.-other affiliation
Ryan S.-other affiliation
Adamson Antony D.-other affiliation
Harper Claire V.V.-University of Manchester (GB)
Bridge L.-other affiliation
Spiller David G.-other affiliation
Jackson Dean A.-other affiliation
Paszek P.-other affiliation
Sée V.-other affiliation
White Michael R.R.-University of Manchester (GB)
3.Finkenstädt B., Woodcock D.J., Komorowski M., Harper C.V., Davis J.R.E., White M.R.H., Rand D.A., Quantifying intrinsic and extrinsic noise in gene transcription using the linear noise approximation: An application to single cell data, Annals of Applied Statistics, ISSN: 1932-6157, DOI: 10.1214/13-AOAS669, Vol.7, No.4, pp.1960-1982, 2013
Abstract:

A central challenge in computational modeling of dynamic biological systems is parameter inference from experimental time course measurements. However, one would not only like to infer kinetic parameters but also study their variability from cell to cell. Here we focus on the case where single-cell fluorescent protein imaging time series data are available for a population of cells. Based on van Kampen’s linear noise approximation, we derive a dynamic state space model for molecular populations which is then extended to a hierarchical model. This model has potential to address the sources of variability relevant to single-cell data, namely, intrinsic noise due to the stochastic nature of the birth and death processes involved in reactions and extrinsic noise arising from the cell-to-cell variation of kinetic parameters. In order to infer such a model from experimental data, one must also quantify the measurement process where one has to allow for nonmeasurable molecular species as well as measurement noise of unknown level and variance. The availability of multiple single-cell time series data here provides a unique testbed to fit such a model and quantify these different sources of variation from experimental data.

Keywords:

Linear noise approximation, kinetic parameter estimation, intrinsic and extrinsic noise, state space model and Kalman filter, Bayesian hierarchical modeling

Affiliations:
Finkenstädt B.-University of Warwick (GB)
Woodcock D.J.-University of Warwick (GB)
Komorowski M.-IPPT PAN
Harper C.V.-University of Manchester (GB)
Davis J.R.E.-University of Manchester (GB)
White M.R.H.-University of Manchester (GB)
Rand D.A.-University of Warwick (GB)
4.Paszek P., Ryan S., Ashall L., Sillitoe K., Harper C. V., Spiller David G., Rand D. A., White M., Population robustness arising from cellular heterogeneity, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, ISSN: 0027-8424, DOI: 10.1073/pnas.0913798107, Vol.107, No.25, pp.11644-11649, 2010
Abstract:

Heterogeneity between individual cells is a common feature of dynamic cellular processes, including signaling, transcription, and cell fate; yet the overall tissue level physiological phenotype needs to be carefully controlled to avoid fluctuations. Here we show that in the NF-κB signaling system, the precise timing of a dual-delayed negative feedback motif [involving stochastic transcription of inhibitor κB (IκB)-α and -ε] is optimized to induce heterogeneous timing of NF-κB oscillations between individual cells. We suggest that this dual-delayed negative feedback motif enables NF-κB signaling to generate robust single cell oscillations by reducing sensitivity to key parameter perturbations. Simultaneously, enhanced cell heterogeneity may represent a mechanism that controls the overall coordination and stability of cell population responses by decreasing temporal fluctuations of paracrine signaling. It has often been thought that dynamic biological systems may have evolved to maximize robustness through cell-to-cell coordination and homogeneity. Our analyses suggest in contrast, that this cellular variation might be advantageous and subject to evolutionary selection. Alternative types of therapy could perhaps be designed to modulate this cellular heterogeneity.

Affiliations:
Paszek P.-IPPT PAN
Ryan S.-other affiliation
Ashall L.-other affiliation
Sillitoe K.-other affiliation
Harper C. V.-University of Manchester (GB)
Spiller David G.-other affiliation
Rand D. A.-University of Warwick (GB)
White M.-other affiliation
5.Harper Claire V.V., Featherstone K., Semprini S., Friedrichsen S., McNeilly J., Paszek P., Spiller David G., McNeilly Alan S., Mullins John J., Davis Julian R., White Michael R.R., Dynamic organisation of prolactin gene expression in living pituitary tissue, Journal of Cell Science, ISSN: 0021-9533, DOI: 10.1242/jcs.060434, Vol.123, No.3, pp.424-430, 2010
Abstract:

Gene expression in living cells is highly dynamic, but temporal patterns of gene expression in intact tissues are largely unknown. The mammalian pituitary gland comprises several intermingled cell types, organised as interdigitated networks that interact functionally to generate co-ordinated hormone secretion. Live-cell imaging was used to quantify patterns of reporter gene expression in dispersed lactotrophic cells or intact pituitary tissue from bacterial artificial chromosome (BAC) transgenic rats in which a large prolactin genomic fragment directed expression of luciferase or destabilised enhanced green fluorescent protein (d2EGFP). Prolactin promoter activity in transgenic pituitaries varied with time across different regions of the gland. Although amplitude of transcriptional responses differed, all regions of the gland displayed similar overall patterns of reporter gene expression over a 50-hour period, implying overall co-ordination of cellular behaviour. By contrast, enzymatically dispersed pituitary cell cultures showed unsynchronised fluctuations of promoter activity amongst different cells, suggesting that transcriptional patterns were constrained by tissue architecture. Short-term, high resolution, single cell analyses in prolactin-d2EGFP transgenic pituitary slice preparations showed varying transcriptional patterns with little correlation between adjacent cells. Together, these data suggest that pituitary tissue comprises a series of cell ensembles, which individually display a variety of patterns of short-term stochastic behaviour, but together yield long-range and long-term coordinated behaviour.

Keywords:

Live-cell, Microscopy, Pituitary, Prolactin, Transcription

Affiliations:
Harper Claire V.V.-University of Manchester (GB)
Featherstone K.-other affiliation
Semprini S.-other affiliation
Friedrichsen S.-other affiliation
McNeilly J.-other affiliation
Paszek P.-other affiliation
Spiller David G.-other affiliation
McNeilly Alan S.-other affiliation
Mullins John J.-other affiliation
Davis Julian R.-University of Manchester (GB)
White Michael R.R.-University of Manchester (GB)
6.Turner D., Paszek P., Woodcock D. J., Nelson David E., Horton Caroline A., Yunjiao W., Spiller David G., Rand D. A., White M., Harper C. V., Physiological levels of TNFalpha stimulation induce stochastic dynamics of NF-kappaB responses in single living cells, Journal of Cell Science, ISSN: 0021-9533, DOI: 10.1242/jcs.069641, Vol.123, No.16, pp.2834-2843, 2010
Abstract:

Nuclear factor kappa B (NF-kappaB) signalling is activated by cellular stress and inflammation and regulates cytokine expression. We applied single-cell imaging to investigate dynamic responses to different doses of tumour necrosis factor alpha (TNFalpha). Lower doses activated fewer cells and those responding showed an increasingly variable delay in the initial NF-kappaB nuclear translocation and associated IkappaBalpha degradation. Robust 100 minute nuclear:cytoplasmic NF-kappaB oscillations were observed over a wide range of TNFalpha concentrations. The result is supported by computational analyses, which identified a limit cycle in the system with a stable 100 minute period over a range of stimuli, and indicated no co-operativity in the pathway activation. These results suggest that a stochastic threshold controls functional all-or-nothing responses in individual cells. Deterministic and stochastic models simulated the experimentally observed activation threshold and gave rise to new predictions about the structure of the system and open the way for better mechanistic understanding of physiological TNFalpha activation of inflammatory responses in cells and tissues.

Keywords:

NF-

Affiliations:
Turner D.-other affiliation
Paszek P.-IPPT PAN
Woodcock D. J.-University of Warwick (GB)
Nelson David E.-other affiliation
Horton Caroline A.-other affiliation
Yunjiao W.-other affiliation
Spiller David G.-other affiliation
Rand D. A.-University of Warwick (GB)
White M.-other affiliation
Harper C. V.-University of Manchester (GB)
7.Ashall L., Horton Caroline A., Nelson David E., Paszek P., Harper Claire V.V., Sillitoe K., Ryan S., Spiller David G., Unitt John F., Broomhead David S., Kell Douglas B., Rand David A.A., Sée V., White Michael R.R., Pulsatile Stimulation Determines Timing and Specificity of NF-κB-Dependent Transcription, Science, ISSN: 0036-8075, DOI: 10.1126/science.1164860, Vol.324, No.5924, pp.242-246, 2009
Abstract:

The nuclear factor κB (NF-κB) transcription factor regulates cellular stress responses and the immune response to infection. NF-κB activation results in oscillations in nuclear NF-κB abundance. To define the function of these oscillations, we treated cells with repeated short pulses of tumor necrosis factor–α at various intervals to mimic pulsatile inflammatory signals. At all pulse intervals that were analyzed, we observed synchronous cycles of NF-κB nuclear translocation. Lower frequency stimulations gave repeated full-amplitude translocations, whereas higher frequency pulses gave reduced translocation, indicating a failure to reset. Deterministic and stochastic mathematical models predicted how negative feedback loops regulate both the resetting of the system and cellular heterogeneity. Altering the stimulation intervals gave different patterns of NF-κB–dependent gene expression, which supports the idea that oscillation frequency has a functional role.

Affiliations:
Ashall L.-other affiliation
Horton Caroline A.-other affiliation
Nelson David E.-other affiliation
Paszek P.-other affiliation
Harper Claire V.V.-University of Manchester (GB)
Sillitoe K.-other affiliation
Ryan S.-other affiliation
Spiller David G.-other affiliation
Unitt John F.-other affiliation
Broomhead David S.-other affiliation
Kell Douglas B.-other affiliation
Rand David A.A.-University of Warwick (GB)
Sée V.-other affiliation
White Michael R.R.-University of Manchester (GB)