Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters

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.

Timing of gene expression in a cell‐fate decision system

“…Therefore, we wanted to measure, in the mating pathway, how kinase activity and gene expression were temporally correlated. Using fluorescent relocation sensors that we previously engineered, we are able to quantify, in real‐time and at the single‐cell level, both MAPK activity and gene expression upon stimulation of MATa cells with synthetic pheromone (α‐factor, 1 μM; Durandau et al , ; Aymoz et al , ). Signaling activity was quantified using a Ste7 DS ‐SKARS Y , which exits the nucleus when the mating MAPKs Fus3 and Kss1 phosphorylate specific residues in the vicinity of a nuclear localization sequence (NLS) (Fig A; Appendix Fig S2A).…”

“…FIG1 displays the largest fold induction upon pheromone stimulation (Roberts et al , ). In this assay, the FIG1 promoter drives the expression of a small peptide, which interacts with a fluorescent protein and promotes its recruitment in the nucleus (Fig A, Appendix Fig S2B, Aymoz et al , ). Upon stimulation, the cells activate the mating MAPKs a few minutes after stimulation, as previously described (Yu et al , ; Nagiec & Dohlman, ; Durandau et al , ).…”

“…We used a second protein expression reporter, the dPSTR Y , which is orthogonal to the dPSTR R , to quantify p AGA1 and p FIG1 expression dynamics in the same strain (Aymoz et al , ; Fig E and Appendix Fig S3B). In all expressing cells, the response time for each promoter was determined based on the time at which the dPSTR nuclear enrichment reached 20% of its maximum (Fig F, see Materials and Methods and Appendix Fig S4).…”

“…However, the slow maturation time of fluorescent proteins (FP) precluded thorough investigations of gene expression kinetics. In a previous paper, we developed the dPSTR, a fluorescent relocation reporter that converts the expression of a promoter into a signal of relocation of a fluorescent protein (Aymoz et al , ).…”

Chiral Selective Transport of Proteins by Cysteine‐Enantiomer‐Modified Nanopores

“…[1][2][3][4] Chirality is an intriguing and intrinsic feature of life and is extensively found in most biomolecular components of living organisms,s uch as DNA, proteins,p hospholipids,a nd sugars. [1][2][3][4] Chirality is an intriguing and intrinsic feature of life and is extensively found in most biomolecular components of living organisms,s uch as DNA, proteins,p hospholipids,a nd sugars.…”