Linear Approaches to Intramolecular Forster Resonance Energy Transfer Probe Measurements for Quantitative Modeling
AuthorsBirtwistle, Marc R.
von Kriegsheim, Alexander
Schwarz, Juliane P.
Anderson, Kurt I.
MetadataShow full item record
AbstractNumerous unimolecular, genetically-encoded Forster 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 Ralt) 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 Ralt are the preferred techniques for obtaining quantitative data from FRET probe experiments for mathematical modeling purposes.
CitationPLoS One. 2011 Nov 16; 6(11):e27823
- FRET-based small-molecule fluorescent probes: rational design and bioimaging applications.
- Authors: Yuan L, Lin W, Zheng K, Zhu S
- Issue date: 2013 Jul 16
- Fluorescence lifetime readouts of Troponin-C-based calcium FRET sensors: a quantitative comparison of CFP and mTFP1 as donor fluorophores.
- Authors: Laine R, Stuckey DW, Manning H, Warren SC, Kennedy G, Carling D, Dunsby C, Sardini A, French PM
- Issue date: 2012
- In vivo imaging of signal transduction cascades with probes based on Förster Resonance Energy Transfer (FRET).
- Authors: Nakamura T, Matsuda M
- Issue date: 2009 Dec
- Global analysis of Förster resonance energy transfer in live cells measured by fluorescence lifetime imaging microscopy exploiting the rise time of acceptor fluorescence.
- Authors: Laptenok SP, Borst JW, Mullen KM, van Stokkum IH, Visser AJ, van Amerongen H
- Issue date: 2010 Jul 21
- Time-resolved FRET fluorescence spectroscopy of visible fluorescent protein pairs.
- Authors: Visser AJ, Laptenok SP, Visser NV, van Hoek A, Birch DJ, Brochon JC, Borst JW
- Issue date: 2010 Jan
Showing items related by title, author, creator and subject.
Early Development of the Central and Peripheral Nervous Systems Is Coordinated by Wnt and BMP SignalsPatthey, Cédric; Gunhaga, Lena; Edlund, Thomas; Mei, Lin; Department of Neurology (2008-02-20)The formation of functional neural circuits that process sensory information requires coordinated development of the central and peripheral nervous systems derived from neural plate and neural plate border cells, respectively. Neural plate, neural crest and rostral placodal cells are all specified at the late gastrula stage. How the early development of the central and peripheral nervous systems are coordinated remains, however, poorly understood. Previous results have provided evidence that at the late gastrula stage, graded Wnt signals impose rostrocaudal character on neural plate cells, and Bone Morphogenetic Protein (BMP) signals specify olfactory and lens placodal cells at rostral forebrain levels. By using in vitro assays of neural crest and placodal cell differentiation, we now provide evidence that Wnt signals impose caudal character on neural plate border cells at the late gastrula stage, and that under these conditions, BMP signals induce neural crest instead of rostral placodal cells. We also provide evidence that both caudal neural and caudal neural plate border cells become independent of further exposure to Wnt signals at the head fold stage. Thus, the status of Wnt signaling in ectodermal cells at the late gastrula stage regulates the rostrocaudal patterning of both neural plate and neural plate border, providing a coordinated spatial and temporal control of the early development of the central and peripheral nervous systems.
beta-Catenin Regulates Intercellular Signalling Networks and Cell-Type Specific Transcription in the Developing Mouse Midbrain-Rhombomere 1 RegionChilov, Dmitri; Sinjushina, Natalia; Saarimaki-Vire, Jonna; Taketo, Makoto M.; Partanen, Juha; Mei, Lin; Department of Neurology (2010-06-3)b-catenin is a multifunctional protein involved in both signalling by secreted factors of Wnt family and regulation of the cellular architecture. We show that b-catenin stabilization in mouse midbrain-rhombomere1 region leads to robust upregulation of several Wnt signalling target genes, including Fgf8. Suggestive of direct transcriptional regulation of the Fgf8 gene, b-catenin stabilization resulted in Fgf8 up-regulation also in other tissues, specifically in the ventral limb ectoderm. Interestingly, stabilization of b-catenin rapidly caused down-regulation of the expression of Wnt1 itself, suggesting a negative feedback loop. The changes in signal molecule expression were concomitant with deregulation of anteriorposterior and dorso-ventral patterning. The transcriptional regulatory functions of b-catenin were confirmed by b-catenin loss-of-function experiments. Temporally controlled inactivation of b-catenin revealed a cell-autonomous role for b-catenin in the maintenance of cell-type specific gene expression in the progenitors of midbrain dopaminergic neurons. These results highlight the role of b-catenin in establishment of neuroectodermal signalling centers, promoting region-specific gene expression and regulation of cell fate determination.
Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain SensationCanetta, Sarah E.; Luca, Edlira; Pertot, Elyse; Role, Lorna W.; Talmage, David A.; Mei, Lin; Department of Neurology; College of Graduate Studies (2011-09-20)Type III Nrg1, a member of the Nrg1 family of signaling proteins, is expressed in sensory neurons, where it can signal in a bi-directional manner via interactions with the ErbB family of receptor tyrosine kinases (ErbB RTKs)