Linear Approaches to Intramolecular Förster Resonance Energy Transfer Probe Measurements for Quantitative Modeling

University of Hong Kong, Hong Kong
PLoS ONE (Impact Factor: 3.23). 11/2011; 6(11):e27823. DOI: 10.1371/journal.pone.0027823
Source: PubMed


Numerous unimolecular, genetically-encoded Förster Resonance Energy Transfer (FRET) probes for monitoring biochemical activities in live cells have been developed over the past decade. As these probes allow for collection of high frequency, spatially resolved data on signaling events in live cells and tissues, they are an attractive technology for obtaining data to develop quantitative, mathematical models of spatiotemporal signaling dynamics. However, to be useful for such purposes the observed FRET from such probes should be related to a biological quantity of interest through a defined mathematical relationship, which is straightforward when this relationship is linear, and can be difficult otherwise. First, we show that only in rare circumstances is the observed FRET linearly proportional to a biochemical activity. Therefore in most cases FRET measurements should only be compared either to explicitly modeled probes or to concentrations of products of the biochemical activity, but not to activities themselves. Importantly, we find that FRET measured by standard intensity-based, ratiometric methods is inherently non-linear with respect to the fraction of probes undergoing FRET. Alternatively, we find that quantifying FRET either via (1) fluorescence lifetime imaging (FLIM) or (2) ratiometric methods where the donor emission intensity is divided by the directly-excited acceptor emission intensity (denoted R(alt)) is linear with respect to the fraction of probes undergoing FRET. This linearity property allows one to calculate the fraction of active probes based on the FRET measurement. Thus, our results suggest that either FLIM or ratiometric methods based on R(alt) are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.

Download full-text


Available from: Kurt I Anderson
  • Source
    • "This indicates that biosensor expression affects the MAPK signaling network to some extent. Biosensor FRET ratio measurements do not necessarily scale linearly with the signaling events they report on, and it has previously proven valuable to explicitly model this (Birtwistle et al, 2011;Fujita et al, 2014). However, given the strong similarity between the Western blot, immunofluorescence, and EKAR2G datasets, we assumed that FRET ratio measurements could be used directly as a proxy for ERK activity. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Transient versus sustained ERK MAP kinase (MAPK) activation dynamics induce proliferation versus differentiation in response to epidermal (EGF) or nerve (NGF) growth factors in PC-12 cells. Duration of ERK activation has therefore been proposed to specify cell fate decisions. Using a biosensor to measure ERK activation dynamics in single living cells reveals that sustained EGF/NGF application leads to a heterogeneous mix of transient and sustained ERK activation dynamics in distinct cells of the population, different than the population average. EGF biases toward transient, while NGF biases toward sustained ERK activation responses. In contrast, pulsed growth factor application can repeatedly and homogeneously trigger ERK activity transients across the cell population. These datasets enable mathematical modeling to reveal salient features inherent to the MAPK network. Ultimately, this predicts pulsed growth factor stimulation regimes that can bypass the typical feedback activation to rewire the system toward cell differentiation irrespective of growth factor identity.
    Full-text · Article · Nov 2015 · Molecular Systems Biology
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The EGF-stimulated ERK/MAPK pathway is a key conduit for cellular proliferation signals and a therapeutic target in many cancers. Here, we characterize two central quantitative aspects of this pathway: the mechanism by which signal strength is encoded and the response curve relating signal output to proliferation. Under steady-state conditions, we find that ERK is activated in discrete, asynchronous pulses with frequency and duration determined by extracellular concentrations of EGF spanning the physiological range. In genetically identical sister cells, cell-to-cell variability in pulse dynamics influences the decision to enter S phase. While targeted inhibition of EGFR reduces the frequency of ERK activity pulses, inhibition of MEK reduces their amplitude. Continuous response curves measured in multiple cell lines reveal that proliferation is effectively silenced only when ERK pathway output falls below a threshold of ∼10%, indicating that high-dose targeting of the pathway is necessary to achieve therapeutic efficacy.
    Preview · Article · Dec 2012 · Molecular cell