title: Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution.
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.
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California, USA.
authors.forename: Gary C H
authors.initials: GC
authors.lastname: Mo
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California, USA.|Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Ross
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California, USA.
authors.forename: Fabian
authors.initials: F
authors.lastname: Hertel
authors.affiliation: JILA, University of Colorado and NIST, Boulder, Colorado, USA.|Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, Colorado, USA.
authors.forename: Premashis
authors.initials: P
authors.lastname: Manna
authors.affiliation: Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Xinxing
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Greenwald
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California, USA.
authors.forename: Chris
authors.initials: C
authors.lastname: Booth
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: Ashlee M
authors.initials: AM
authors.lastname: Plummer
authors.affiliation: Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Tenner
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Zan
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, USA.
authors.forename: Yuxiao
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, Georgia, USA.
authors.forename: Eileen J
authors.initials: EJ
authors.lastname: Kennedy
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Philip A
authors.initials: PA
authors.lastname: Cole
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, USA.
authors.forename: Karen G
authors.initials: KG
authors.lastname: Fleming
authors.affiliation: Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, Colorado, USA.|BioFrontiers Institute, University of Colorado, Boulder, Boulder, Colorado, USA.
authors.forename: Amy
authors.initials: A
authors.lastname: Palmer
authors.affiliation: JILA, University of Colorado and NIST, Boulder, Colorado, USA.|Department of Chemistry and Biochemistry, University of Colorado, Boulder, Boulder, Colorado, USA.
authors.forename: Ralph
authors.identifier: http://orcid.org/0000-0002-8989-405X
authors.initials: R
authors.lastname: Jimenez
authors.affiliation: Department of Biophysics and Biophysical Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Xiao
authors.affiliation: Department of Chemistry, KU Leuven, Heverlee, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Dedecker
authors.affiliation: Department of Pharmacology, University of California San Diego, La Jolla, California, USA.|Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors.forename: Jin
authors.initials: J
authors.lastname: Zhang
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008853:Microscopy / Q000295:instrumentation / Q000379:methods; D057054:Molecular Imaging / Q000379:methods; D016297:Mutagenesis, Site-Directed; D025941:Protein Interaction Mapping / Q000379:methods; D013269:Stochastic Processes
chemical_list: D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D017868:Cyclic AMP-Dependent Protein Kinases