CAST: An automated segmentation and tracking tool for the analysis of transcriptional kinetics from single-cell time-lapse recordings

Blanchoud, Simon; Nicolas, Damien; Zoller, Benjamin; Tidin, Onur; Naef, Félix

doi:10.1016/j.ymeth.2015.04.023

Cited by 9 publications

(11 citation statements)

References 72 publications

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“…The circadian clock was synchronized with dexamethasone before recording with an Actimetrics LumiCycle 32 (population) or LuminoView LV200 microscope (single-cell). Single-cell recordings were analyzed with the CAST platform ( 49 ).…”

Section: Methodsmentioning

confidence: 99%

Modulation of transcriptional burst frequency by histone acetylation

Nicolas

Zoller

Suter

et al. 2018

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

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149

147

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SignificanceSingle-cell approaches have shown that many mammalian genes are transcribed stochastically in bursts of specific sizes and frequencies; however, molecular mechanisms controlling these bursting parameters have remained largely undetermined. By studying transcriptional bursting of a luciferase reporter controlled by a circadian gene promoter, we found that the gene integration site mainly influenced the burst size, while the circadian time primarily modulated the burst frequency. These daily variations in burst frequency correlated with histone acetylation levels, and CRISPR-Cas9–mediated acetylation of the promoter was sufficient to change the burst frequency. Since this correlation was also observed in other genes and in several cell types, we conclude that the impact of histone acetylation on gene expression is achieved mainly through modulation of burst frequency.

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Section: Methodsmentioning

confidence: 99%

Modulation of transcriptional burst frequency by histone acetylation

Nicolas

Zoller

Suter

et al. 2018

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

Self Cite

149

147

View full text Add to dashboard Cite

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“…For higher numbers of cells, a more automated approach should be considered. Various automated tools have been developed in the past years, which allow for segmenting and tracking larger numbers of cells 16,17 . However, the movement of the cells and frequent cell divisions are challenging and prone to cause segmentation errors.…”

Section: Discussionmentioning

confidence: 99%

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Alber

Suter

2018

JoVE

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Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

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“…Cell division and cell death can be well investigated with TLM [50,52,[95][96][97]. Division and growth of both labeled [96,[98][99][100] and non-labeled [101,102] cells in culture [52,95,98,[103][104][105] and tissue slices [106], including monitoring of a single cell [95,99,[107][108][109][110][111][112], can be observed and assessed with TLM. The fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can effectively label individual G1, S/G2/M and G1/S-transition phase nuclei as red, green and yellow, respectively, to visualize the real-time cell cycle transitions in living mammalian cells [113][114][115][116].…”

Section: Time-lapse Microscopy: From Making Movies To Bedside 21 Vementioning

confidence: 99%

“…By now, various algorithms are designed for quantifying and tracking cell migration [3] and Time-Lapse Microscopy http://dx.doi.org/10.5772/intechopen.81199 single cell motility [261,263]; cell proliferation [264]; cell cycle and cell lineage analysis [107]; changes in mitotic and interphase duration [141]; cell-cell contacts [52]; studying specific cells and tissues [265] and specific intracellular processes such as transcription [99] or morphogenesis [266]; colocalization of cells and intracellular markers [184]; tracking cellular organelles [258]; highlighting the certain cell type within tissues or mixed cell cultures [267]; clustered, overlapping or dying cells [268]; in toto imaging of developing organisms, tissues and organs [241] and assessing development and selection of embryos for in vitro fertilization [269,270].…”

Section: Tlm Technical Approachesmentioning

confidence: 99%