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Nat Methods. 2017 Apr;14(4):427-434. doi: 10.1038/nmeth.4221. Epub 2017 Mar 13.

Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.

Author information

1
Department of Pharmacology, University of California San Diego, La Jolla, California, USA.
2
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA.
3
JILA, University of Colorado and NIST, Boulder, Colorado, USA.
4
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, Colorado, USA.
5
Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
6
T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
7
Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
8
Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, USA.
9
BioFrontiers Institute, University of Colorado, Boulder, Boulder, Colorado, USA.
10
Department of Chemistry, KU Leuven, Heverlee, Belgium.

Abstract

Compartmentalized biochemical activities are essential to all cellular processes, but there is no generalizable method to visualize dynamic protein activities in living cells at a resolution commensurate with cellular compartmentalization. Here, we introduce a new class of fluorescent biosensors that detect biochemical activities in living cells at a resolution up to threefold better than the diffraction limit. These 'FLINC' biosensors use binding-induced changes in protein fluorescence dynamics to translate kinase activities or protein-protein interactions into changes in fluorescence fluctuations, which are quantifiable through stochastic optical fluctuation imaging. A protein kinase A (PKA) biosensor allowed us to resolve minute PKA activity microdomains on the plasma membranes of living cells and to uncover the role of clustered anchoring proteins in organizing these activity microdomains. Together, these findings suggest that biochemical activities of the cell are spatially organized into an activity architecture whose structural and functional characteristics can be revealed by these new biosensors.

PMID:
28288122
PMCID:
PMC5388356
DOI:
10.1038/nmeth.4221
[Indexed for MEDLINE]
Free PMC Article

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