Non-Invasive Interogation of Signaling Activated Gene Regulation

Abstract

Many signal transduction and gene regulatory pathways are highly dynamic resulting in a variety of dynamic signaling and gene expression profiles. Currently, our biophysical understanding of these profiles relies on the manipulation of specific genes or drug treatment of specific proteins. One drawback of this approach is that a gene of interest needs to be identified to affect the pathways dynamic. Another drawback is that such perturbations may result in a significant interference with the function of the cell. In order to avoid these drawbacks, we developed a novel non-invasive perturbation approach to investigate the dynamic properties of signaling and gene regulatory pathways, without genetic manipulation or drug treatments. To demonstrate the feasibility of this approach we choose to interrogate a stress response pathway in yeast, which enables us to manipulate the intensity, duration and shape of the signal transduction profile. By combining quantitative single cell and single molecule experiments with predictive modeling, we are able to quantify signal transduction activation, signal transduction saturation and gene expression activation thresholds, which is not possible to quantify with any other currently available technology. We also found that the signaling dynamics is proportional to the first time derivative of the external perturbation profile. Because this approach is independent of the biological pathway or organism, it presents a general methodology to interrogate signaling and gene expression pathways non-invasively without the need for genetic or drug perturbations.