Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 110, Issue 1, Pages 135-140Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1211882110
Keywords
cellular diffusion; fluorescence correlation spectroscopy
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Funding
- Cell Migration Consortium Grants [U54 GM064346]
- [NIH-P41 P41-RRO3155]
- [NIH-P50-GM076516]
- [GM090317]
- [GM057464]
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Here we present a fluctuation-based approach to biosensor Forster 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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