Claire V. Harper

Pulsatile Stimulation Determines Timing and Specificity of NF-κB-Dependent Transcription

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.

Dynamic Analysis of Stochastic Transcription Cycles

Cycling of gene expression in individual cells is controlled by dynamic chromatin remodeling.

Population robustness arising from cellular heterogeneity

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.

Physiological levels of TNFα stimulation induce stochastic dynamics of NF-κB responses in single living cells

Nuclear factor kappa B (NF-κB) 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 (TNFα). Lower doses activated fewer cells and those responding showed an increasingly variable delay in the initial NF-κB nuclear translocation and associated IκBα degradation. Robust 100 minute nuclear:cytoplasmic NF-κB oscillations were observed over a wide range of TNFα 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 TNFα activation of inflammatory responses in cells and tissues.

Ca2+ signalling in the control of motility and guidance in mammalian sperm.

Ca2+ signalling in the sperm plays a key role in the regulation of events preceding fertilisation. Control of motility, including hyperactivation and chemotaxis, is particularly dependent upon [Ca2+]i signalling in the principal piece of the flagellum and the midpiece. Here we briefly review the processes that contribute to regulation of [Ca2+]i in mammalian sperm and then examine two areas: (i) the regulation of hyperactivation by [Ca2+]i and the pivotal roles played by CatSpers (sperm-specific, Ca2+-permeable membrane channels) and intracellular Ca2+ stores in this process and (ii) the elevation of [Ca2+]i and consequent modulation of motility caused by progesterone including the ability of progesterone at micromolar concentrations to cause sperm hyperactivation and/or accumulation and the recent discovery that progesterone, at picomolar concentrations, acts as a chemoattractant for mammalian sperm..

Bayesian inference of biochemical kinetic parameters using the linear noise approximation

BackgroundFluorescent and luminescent gene reporters allow us to dynamically quantify changes in molecular species concentration over time on the single cell level. The mathematical modeling of their interaction through multivariate dynamical models requires the deveopment of effective statistical methods to calibrate such models against available data. Given the prevalence of stochasticity and noise in biochemical systems inference for stochastic models is of special interest. In this paper we present a simple and computationally efficient algorithm for the estimation of biochemical kinetic parameters from gene reporter data.ResultsWe use the linear noise approximation to model biochemical reactions through a stochastic dynamic model which essentially approximates a diffusion model by an ordinary differential equation model with an appropriately defined noise process. An explicit formula for the likelihood function can be derived allowing for computationally efficient parameter estimation. The proposed algorithm is embedded in a Bayesian framework and inference is performed using Markov chain Monte Carlo.ConclusionThe major advantage of the method is that in contrast to the more established diffusion approximation based methods the computationally costly methods of data augmentation are not necessary. Our approach also allows for unobserved variables and measurement error. The application of the method to both simulated and experimental data shows that the proposed methodology provides a useful alternative to diffusion approximation based methods.

Secretory pathway Ca(2+)-ATPase (SPCA1) Ca(2)+ pumps, not SERCAs, regulate complex [Ca(2+)](i) signals in human spermatozoa.

The sarcoplasmic-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibitors thapsigargin (0.1-1 μM) and cyclopiazonic acid (10 μM), failed to affect resting [Ca2+] in human spermatozoa. Slow progesterone-induced [Ca2+ i]i oscillations in human spermatozoa, which involve cyclic emptying-refilling of an intracellular Ca2+ store were also insensitive to these inhibitors. Non-selective doses of thapsigargin (5-30 μM, 50-300 times the saturating dose for SERCA inhibition), caused elevation of resting [Ca2+]i and partial, dose-dependent disruption of oscillations. A 10-40 μM concentration of bis(2-hydroxy-3-tert-butyl-5-methyl-phenyl)methane (bis-phenol), which inhibits both thapsigargin-sensitive and -insensitive microsomal Ca2+ ATPases, caused elevation of resting [Ca2+]i and inhibition of [Ca2+]i oscillations at doses consistent with inhibition of thapsigargin-resistant, microsomal ATPase and liberation of stored Ca2+. Low doses of bis-phenol had marked effects on [Ca2+]i oscillation kinetics. Application of the drug to cells previously stimulated with progesterone had effects very similar to those observed when it was applied to unstimulated cells, suggesting that the sustained Ca2+ influx induced by progesterone is not mediated via mobilisation of Ca2+ stores. Western blotting for human sperm proteins showed expression of secretory pathway Ca2+ ATPase (SPCA1). Immunolocalisation studies revealed expression of SPCA1 in all cells in an area behind the nucleus, extending into the midpiece. Staining for SERCA, carried out in parallel, detected no expression with either technique. We conclude that: (1) intracellular Ca2+ store(s) and store-dependent [Ca2+]i oscillations in human spermatozoa rely primarily on a thapsigargin/cyclopiazonic acid-insensitive Ca2+ pump, which is not a SERCA as characterised in somatic cells; (2) effects of high-dose thapsigargin on spermatozoa primarily reflect non-specific actions on non-SERCAs and; (3) secretory pathway Ca2+ ATPases contribute at least part of this non-SERCA Ca2+ pump activity.

Reconstruction of transcriptional dynamics from gene reporter data using differential equations

Motivation: Promoter-driven reporter genes, notably luciferase and green fluorescent protein, provide a tool for the generation of a vast array of time-course data sets from living cells and organisms. The aim of this study is to introduce a modeling framework based on stochastic differential equations (SDEs) and ordinary differential equations (ODEs) that addresses the problem of reconstructing transcription time-course profiles and associated degradation rates. The dynamical model is embedded into a Bayesian framework and inference is performed using Markov chain Monte Carlo algorithms. Results: We present three case studies where the methodology is used to reconstruct unobserved transcription profiles and to estimate associated degradation rates. We discuss advantages and limits of fitting either SDEs ODEs and address the problem of parameter identifiability when model variables are unobserved. We also suggest functional forms, such as on/off switches and stimulus response functions to model transcriptional dynamics and present results of fitting these to experimental data. Contact: b.f.finkenstadt@warwick.ac.uk Supplementary Information: Supplementary data are available at Bioinformatics online.

Dynamic resolution of acrosomal exocytosis in human sperm.

An essential step in mammalian fertilisation is the sperm acrosome reaction (AR) – exocytosis of a single large vesicle (the acrosome) that surrounds the nucleus at the apical sperm head. The acrosomal and plasma membranes fuse, resulting in both the release of factors that might facilitate penetration of the zona pellucida (which invests the egg) and the externalisation of membrane components required for gamete fusion. Exocytosis in somatic cells is a rapid process – typically complete within milliseconds – yet acrosomal enzymes are required throughout zona penetration – a period of minutes. Here, we present the first studies of this crucial and complex event occurring in real-time in individual live sperm using time-lapse fluorescence microscopy. Simultaneous imaging of separate probes for acrosomal content and inner acrosomal membrane show that rapid membrane fusion, initiated at the cell apex, is followed by exceptionally slow dispersal of acrosomal content (up to 12 minutes). Cells that lose their acrosome prematurely (spontaneous AR), compromising their ability to penetrate the egg vestments, are those that are already subject to a loss of motility and viability. Cells undergoing stimulus-induced AR (progesterone or A23187) remain viable, with a proportion remaining motile (progesterone). These findings suggest that the AR is a highly adapted form of exocytosis.

Dynamic organisation of prolactin gene expression in living pituitary tissue

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.