Stochastic protein expression in individual cells at the single molecule level. Cai, Friedman, Xie in Nature March 16, 2006.
Stochasticity in gene expression is a well known phenomenon, and mechanisms by which cells attenuate stochastic fluxuations to achieve deterministic outcomes is a topic of current research. Here for the first time, Cai, Friedman and Xie characterize the magnitude and variance in single expression events for the beta-galactosidase gene in E. coli and S. cerevisiae. This is achieved using a microfluidic device in which single or very few cells are trapped in 100 picoliter chambers, such that the fluorescent product of beta-gal-catalyzed cleavage of FDG can not diffuse away after being exported from the cell.
By measuring the fluorescence of these chambers using indirect laser excitation, the authors observe discrete changes in the slope of fluorescent intensity over time. The number of beta-gal molecules is then calculated from the time derivative of the fluorescence (compensating for photobleaching) using a calibration factor determined from a previous experiment in which the authors correlate specific values of the intensity slope to integer numbers of beta-gal enzymes.
The authors find that the number of beta-gal enzymes produced in E. coli per expression event follows a decaying exponential distribution, in agreement with the theoretically predicted lifetime of the mRNA molecule. The average number of beta-gal expression events per cell cycle under repressed lacZ conditions was observed to be 0.11, and the average number of beta-gal monomers per event was observed to be 20 (or 5 functional tetramers), also in agreement with previous biochemical estimates. Making the assumption that protein copy numbers are halved at each cell division, this low number of expression events per cell cycle was theoretically shown to give rise to a heterogeneous “all-or-none” population, in which some cells would have copies of the beta-gal enzyme and Lac permease (expressed on the same operon) and thus would be responsive to rises in lactose concentration, whereas other cells would not. This itself is a known and previously unexplained phenomenon.
The only thing I can take away from this paper is that the authors’ method relies on a enzyme-catalyzed cleavage of a fluorescent substrate in order to amplify the fluorescent signal. Thus it is not generalizable to other gene products, although the method can be used to characterize a particular promoter. Hopefully, Cai et al.’s new method will open up research into how regulatory mechanisms can reliably maintain small copy counts of molecules essential for, e.g., signaling in mammalian systems.
*Thanks to Paul L for reporting on this interesting article