Automated tracking of gene expression in individual cells and cell compartments

Shen, Hailin; Nelson, Glyn; Nelson, David E.; Kennedy, Stephnie; Spiller, David G.; Griffiths, Tony; Paton, Norman W.; Oliver, Stephen G.; White, Mike; Kell, Douglas B.

doi:10.1098/rsif.2006.0137

Cited by 56 publications

(49 citation statements)

References 26 publications

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“…Cells were treated with 10 ng/mL of TNFα or IL-1β, and 16 μM of PMA 24 h posttransfection. CellTracker version 0.6 was used for data extraction (42). For RelA fusion proteins, mean fluorescence intensities were calculated for each time point for both nuclei and cytoplasm then nuclear:cytoplasmic (N:C) fluorescence intensity ratios were determined.…”

Section: Methodsmentioning

confidence: 99%

Population robustness arising from cellular heterogeneity

Paszek

Ryan

Ashall

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

176

210

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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.network topology | NF-κB | biological oscillations | feedback regulation | paracrine signaling

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Section: Methodsmentioning

confidence: 99%

Population robustness arising from cellular heterogeneity

Paszek

Ryan

Ashall

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

176

210

View full text Add to dashboard Cite

show abstract

“…Cells were treated with the appropriate stimulation 30 minutes following the onset of time-lapse imaging and images captured every 5 minutes for at least 5 hours. Data were analysed using CellTracker (Shen et al, 2006) by exporting mean fluorescence ratios from the whole of the cytoplasmic and nuclear boundaries for each cell and expressing this as a nuclear to cytoplasmic (N:C) ratio. The average and standard deviation of the N:C ratio from the 30 minute pre-stimulation period were calculated and a threshold set which was two standard deviations above pre-stimulation average; an NF-B response was characterised as being a transient rise in N:C ratio with more than three data points above this threshold (Jameson et al, 2009).…”

Section: Fluorescence Microscopymentioning

confidence: 99%

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

Turner¹,

Paszek²,

Woodcock

et al. 2010

Journal of Cell Science

102

110

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

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“…Average instrument dark count (corrected for the number of pixels being used) was subtracted from the luminescence signal. Timecourse data from fluorescent pituitary slices was analysed using Cell Tracker v0.6 software (Shen et al, 2006).…”

Section: Analysis Of Real-time Imaging Datamentioning

confidence: 99%

Dynamic organisation of prolactin gene expression in living pituitary tissue

Harper

Featherstone

Semprini

et al. 2010

Journal of Cell Science

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SummaryGene 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 coordination 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.

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Automated tracking of gene expression in individual cells and cell compartments

Cited by 56 publications

References 26 publications

Population robustness arising from cellular heterogeneity

Population robustness arising from cellular heterogeneity

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

Dynamic organisation of prolactin gene expression in living pituitary tissue

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