Quantitative relationships between SMAD dynamics and target gene activation kinetics in single live cells.

The transduction of extracellular signals through signaling pathways that culminate in a transcriptional response is central to many biological processes. However, quantitative relationships between activities of signaling pathway components and transcriptional output of target genes remain poorly explored. Here we developed a dual bioluminescence imaging strategy allowing simultaneous monitoring of nuclear translocation of the SMAD4 and SMAD2 transcriptional activators upon TGF-ß stimulation, and the transcriptional response of the endogenous connective tissue growth factor (ctgf) gene. Using cell lines allowing to vary exogenous SMAD4/2 expression levels, we performed quantitative measurements of the temporal profiles of SMAD4/2 translocation and ctgf transcription kinetics in hundreds of individual cells at high temporal resolution. We found that while nuclear translocation efficiency had little impact on initial ctgf transcriptional activation, high total cellular SMAD4 but not SMAD2 levels increased the probability of cells to exhibit a sustained ctgf transcriptional response. The approach we present here allows time-resolved single cell quantification of transcription factor dynamics and transcriptional responses and thereby sheds light on the quantitative relationship between SMADs and target gene responses.

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

Fluorescence and bioluminescence time-lapse imaging allows to investigate a vast range of cellular processes at single-cell or even subcellular resolution. In particular, time-lapse imaging can provide uniquely detailed information on the fine kinetics of transcription, as well as on biological oscillations such as the circadian and cell cycles. However, we face a paucity of automated methods to quantify time-lapse imaging data with single-cell precision, notably throughout multiple cell cycles. We developed CAST (Cell Automated Segmentation and Tracking platform) to automatically and robustly detect the position and size of cells or nuclei, quantify the corresponding light signals, while taking into account both cell divisions (lineage tracking) and migration events. We present here how CAST analyzes bioluminescence data from a short-lived transcriptional luciferase reporter. However, our flexible and modular implementation makes it easily adaptable to a wide variety of time-lapse recordings. We exemplify how CAST efficiently quantifies single-cell gene expression over multiple cell cycles using mouse NIH3T3 culture cells with a luminescence expression driven by the Bmal1 promoter, a central gene of the circadian oscillator. We further illustrate how such data can be used to quantify transcriptional bursting in conditions of lengthened circadian period, revealing thereby remarkably similar bursting signature compared to the endogenous circadian condition despite marked period lengthening. In summary, we establish CAST as novel tool for the efficient segmentation, signal quantification, and tracking of time-lapse images from mammalian cell culture.