Cautionary note on FRET
Some surprising and important news from Nature Biotechnology about a common technique in cellular imagining, fluorescence resonance energy transfer, or FRET. Specifically, it looks like ATP/Mg can significantly alter the FRET signal, which has commonly been used for looking at Ca, voltage, and various other binding interactions in neurons:
Given these findings, we predict that fluctuations in free or Mg2+-bound ATP will affect the signal output of most—if not all—CFP-YFP–based FRET indicators.
The authors continue with the following recommendations:
Currently, not much is known about effects of intracellular molecular crowding or the occurrence of local fluctuations in ATP concentration in distinct cellular locales, including the unstirred molecular layers in the vicinity of membranes8, 9. We think, however, that the dynamic concentration(s) of compartmentalized local free Mg-ATP will almost certainly overlap the 1–20 mM range, or be even higher. Thus, our observations may have important implications for microscopy studies of a broad range of local signaling events or metabolite changes in a broad range of cells. Findings should be interpreted with caution if genetically encoded CFP-YFP–based biosensors are used in cells with large and sudden fluctuations in local or global ATP concentration and appropriate controls with bound and nonbound ligands and CFP and YFP placed at appropriate spacing should be used at all times.
Source: ATP and FRET—a cautionary note
Marieke Willemse, et al. (Feb. 2007)
March 6th, 2007 at 4:38 am
ATP and FRET – A cautionary note, confirmed?
In the February issue of Nature Biotechnology, a correspondence piece noted some very surprising findings regarding the sensitivity of genetically-encoded fluorescence resonance energy transfer (FRET) indicators to adenosine triphosphate.
During the …
March 6th, 2007 at 4:52 am
While I was at first quite skeptical of the reported results, my attitude has changed after trying to replicate their results. Applying ATP to purified GluSnFR gave very similar quenching effects. The authors of this study may be onto something significant. Or we all could be missing an obscure artifact. For a more complete breakdown of the paper and to see some new data supporting their results, check out my weblog post.