Article

Millisecond Spatiotemporal Dynamics of FRET Biosensors by the Pair Correlation Function and the Phasor Approach to FLIM

Laboratory for Fluorescence Dynamics, Department of Biomedical Engineering, and Department of Development and Cell Biology, University of California, Irvine, Irvine, CA 92617.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 12/2012; 110(1). DOI: 10.1073/pnas.1211882110
Source: PubMed

ABSTRACT

Here we present a fluctuation-based approach to biosensor Förster resonance energy transfer (FRET) detection that can measure the molecular flow and signaling activity of proteins in live cells. By simultaneous use of the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and cross-pair correlation function (pCF) analysis along a line scanned in milliseconds, we detect the spatial localization of Rho GTPase activity (biosensor FRET signal) as well as the diffusive route adopted by this active population. In particular we find, for Rac1 and RhoA, distinct gradients of activation (FLIM-FRET) and a molecular flow pattern (pCF analysis) that explains the observed polarized GTPase activity. This multiplexed approach to biosensor FRET detection serves as a unique tool for dissection of the mechanism(s) by which key signaling proteins are spatially and temporally coordinated.

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    • "Additionally, combinations of different lifetimes follow simple vectorial addition rules, allowing for the calculation of the intensity fraction due to each component. Several groups, including us, applied successfully the phasor approach to FLIM to follow a broad range of intracellular processes including biomolecular interactions [17] and ion homeostasis [18]. "
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