Real-time quantification of protein expression at the single-cell level via dynamic protein synthesis translocation reporters.
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.
Department of Fundamental Microbiology, University of Lausanne, Lausanne CH-1015, Switzerland.
Delphine
D
Aymoz
Department of Fundamental Microbiology, University of Lausanne, Lausanne CH-1015, Switzerland.
Victoria
V
Wosika
Department of Fundamental Microbiology, University of Lausanne, Lausanne CH-1015, Switzerland.
Eric
E
Durandau
Department of Fundamental Microbiology, University of Lausanne, Lausanne CH-1015, Switzerland.
Serge
http://orcid.org/0000-0002-0245-049X
S
Pelet
D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D005990:Glycerol / Q000378:metabolism; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+) / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods; D013997:Time Factors; D000097573:Red Fluorescent Protein
D005990:Glycerol; D050536:Glycerol-3-Phosphate Dehydrogenase (NAD+); D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D029701:Saccharomyces cerevisiae Proteins; D000097573:Red Fluorescent Protein; C470555:STL1 protein, S cerevisiae; C568531:GPD1 protein, S cerevisiae