Superfund Research Program

Novel Cell-Based Toxicity Sensors Identified by Genome-Wide Screens

Project Leader: Roger Y. Tsien
Grant Number: P42ES010337
Funding Period: 2000-2005

Project-Specific Links

Final Progress Reports

Year:   2004 

Colette Dooley, a graduate student supported by the Training Core and working under Project 3, has characterized redox indicators consisting of GFP mutants with cysteine pairs, whose reversible oxidation to a disulfide profoundly shifts the excitation spectrum to shorter wavelengths, enabling ratiometric measurement.  She applied these indicators in live cells, targeted them to cytosol, plasma membrane, and nucleus, and increased their oxidation sensitivity by placing positive charges next to the cysteines to lower the pK_a of the latter.  The redox indicators become substantially oxidized in response to exogenous oxidants or oxidative bursts in HL60 cells.  Despite considerable effort, she has not found responses to growth factor stimulation, suggesting that if reactive oxygen species (ROS) are as important in mitogenic signaling as widely believed, such ROS must be very transient or localized .  This work appeared as Dooley, C.M., Dore, T.M., Hanson, G.T., Jackson, W.C., Remington, S.J., and Tsien, R.Y. 2004.  Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators.  J. Biol. Chem. 279: 22284-22293. 

Most recently, Colette fused the redox indicators directly to the EGF receptor, which is supposed to initiate ROS generation, yet saw no oxidation.  Separately she has replaced one or both cysteines by selenocysteine (SeCys) to increase redox sensitivity further.  Incorporation of SeCys into the middle of a protein expressed in mammalian cells is already a significant achievement.  SeCys mutants increase peroxide sensitivity in single cells, but expression at higher levels needed for in vitro biochemistry remains problematic.  Nevertheless the SeCys versions likewise fail to detect ROS during mitogenic stimulation.  The most conclusive proof would be to fuse the redox indicators to PTP-1B, the protein for which there is the most evidence for irreversible oxidation by ROS.  If the redox indicators are still unaffected by mitogenic signaling, that will prove that ROS do not significantly shift the thiol-disulfide reversible equilibrium even locally.  Colette is also trying to design GFP-based indicators that will trap ROS less reversibly.