fluorescent timers: a new biophotonic tool

ResearchBlogging.org
In standard reporter assays, the basal activity of the cloned promoter often results in accumulation of both luciferase mRNA and protein. This “background” activity may be an advantage since, once measured, gives you some numbers that you may use to calculate a fold induction (you can't divide by 0, so you need a basal number). However, accumulation is synonym of slow clearance rate: having in the assay some pre-existing reporter molecules may delay and dilute the measurable response, so that it will be more difficult to obtain accurate quantifications of changes in cell signaling pathways. Hence, in standard assays, transient or relatively minor effects may be hidden and the kinetics somewhat inaccurate: this explains why researchers prefer reporters with short half-life, and eventually decrease it with both mRNA and protein destabilizing elements (see for instance the RapidReporter Gaussia luciferase from Activemotif, or the RapidResponse luc2P from Promega).


Today, another step in the race toward the 4th dimension (time) have been made: new fluorescent 'timers' (FTs) that gradually change color from blue to red could allow researchers to track the age and dynamic behaviour of proteins in living cells. Previous work suggested that some red fluorescent proteins start out fluorescing blue, but then change to red as the protein is chemically modified over time. Vladislav Verkhusha and his colleagues mutated the red fluorescent mCherry toward altered maturation rates from blue to red, getting three fluorescent proteins, each with a specific maturation rate. The proteins were used to track newly synthesized proteins in mammalian cells grown in culture, but maybe in future we will have a "flight recorder" inside a cell, to log different molecular events with respect to time.

Indeed, 'FT' not only means Fluorescent Timer but also Fourier Transform, a mathematical approach you can use among other things, to get information about periodicity in temporal dynamics. Really a good name for a reporter assay giving us more background about temporal events. 

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Fedor V Subach, Oksana M Subach, Illia S Gundorov, Kateryna S Morozova, Kiryl D Piatkevich, Ana Maria Cuervo, Vladislav V Verkhusha (2009). Monomeric fluorescent timers that change color from blue to red report on cellular trafficking Nature Chemical Biology, 5 (2), 118-126 DOI: 10.1038/nchembio.138