Partnership for the Advancement of Cancer Research Project
Cells make key decisions: to proliferate or not, to undergo apoptosis or not, in response to information transmitted through cell signaling systems. Many disorders including cancer can be due to alterations in cell signaling function and components. Operation of cell signaling systems is often best studied by single cell quantification of signals from fluorescent protein reporters. We and others now perform such quantification by image microscopy. Even when automated, these methods are not high throughput. New microscopic methods promise better measurements but no higher throughput. We will thus extend an early-stage collaboration to develop flow cytometry methods to quantify excited state dynamics, fluorescence decays, and average fluorescence lifetimes. For this work, we modulate the excitation light from lasers at a radio frequency. We infer the relaxation rates of fluorescent proteins and other fluorophores from phase-shifts of modulated emission signals relative to Rayleigh scattering signal.
We will use these Time Resolved Flow Cytometry (TRFC) methods to measure cell signaling events, and to extend the reach of FRET reporters to allow new kinds of measurements when the fluorophore finds itself in new environments such as bound to DNA or oligomerized into large protein complexes. We will use these methods to sort cells into subpopulations and use those populations that to determine causes of cell-to-cell variation. Progress will depend on the interplay between measurements of excited state changes of fluorescent protein fluorophores engineered into cells and research on instrumentation to push the resolution limits of phase-containing multiparametric data.
In particular, we will pursue two aims. First, we will develop TFRC and use it to quantify two widely conserved and important classes of signaling events, dissociation of heterotrimeric G proteins, and derepression of DNA bound transcription activators. We will attempt to measure other important, prototypic signaling events (including recruitment of a scaffold protein to the cell membrane, translocation of proteins to the cell nucleus, and binding to regulatory sites on cell DNA). Second, we will develop the ability to sort subpopulations of cells based on differences in TFRC signals. We will use these abilities to undertake a census of system proteins to determine explanations for differences among differently signaling cell populations as above. Progress toward these goals would by definition constitute important science and will position the collaborators to attract nationally competitive funding to continue the work.
To learn more about the research talking place in Dr. Brent’s lab, visit: http://labs.fhcrc.org/brent/index.html
Information about Dr. Houston' research can be found at: http://chemeng.nmsu.edu/JPH/index.html