Abstract
Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. It is widely appreciated that single cells can display large variations in the level of gene induction. However, the variability in the dynamics of this process in individual cells is difficult to quantify using standard fluorescent protein (FP) expression assays, due to the slow maturation of their fluorophore. Here we have developed expression reporters that accurately measure both the levels and dynamics of protein synthesis in live single cells with a temporal resolution under a minute. Our system relies on the quantification of the translocation of a constitutively expressed FP into the nucleus. As a proof of concept, we used these reporters to measure the transient protein synthesis arising from two promoters responding to the yeast hyper osmolarity glycerol mitogen-activated protein kinase pathway (pSTL1 and pGPD1). They display distinct expression dynamics giving rise to strikingly different instantaneous expression noise.
Highlights
Protein expression is a dynamic process, which can be rapidly induced by extracellular signals
We report here on the development and validation of the dynamic protein synthesis translocation reporter, that provides real-time measurement of protein expression arising from a promoter element in live single cells
0.1 M and slightly drops at 0.4 M (Supplementary Fig. 3c–e). This difference could be explained by a saturation effect of the nuclear enrichment of the sensor (Supplementary Note 1 and Supplementary Fig. 4). These results demonstrate that the relocation of the fluorescent protein (FP) in the dynamic protein synthesis translocation reporter (dPSTR) assay provides a real-time measurement of protein synthesis in live single cells, allowing accurate quantification of both levels and kinetics of protein expression arising from a promoter of interest
Summary
Protein expression is a dynamic process, which can be rapidly induced by extracellular signals. We report here on the development and validation of the dynamic protein synthesis translocation reporter (dPSTR), that provides real-time measurement of protein expression arising from a promoter element in live single cells.
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