title: New alternately colored FRET sensors for simultaneous monitoring of Zn²⁺ in multiple cellular locations.
abstract: Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the development of new sensors for Zn²⁺based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn²⁺ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn²⁺ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn²⁺. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn²⁺ uptake simultaneously in two compartments, revealing that nuclear Zn²⁺ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn²⁺ more slowly and with a delay of 600-700 sec. Lastly, these studies provide the first glimpse of nuclear Zn²⁺ and reveal that nuclear Zn²⁺ is buffered at a higher level than cytosolic Zn²⁺.
authors.affiliation: Department of Chemistry and Biochemistry and BioFrontiers Institute, University of Colorado, Boulder, Colorado, United States of America.
authors.forename: Jose G
authors.initials: JG
authors.lastname: Miranda
authors.forename: Amanda L
authors.initials: AL
authors.lastname: Weaver
authors.forename: Yan
authors.initials: Y
authors.lastname: Qin
authors.forename: J Genevieve
authors.initials: JG
authors.lastname: Park
authors.forename: Caitlin I
authors.initials: CI
authors.lastname: Stoddard
authors.forename: Michael Z
authors.initials: MZ
authors.lastname: Lin
authors.forename: Amy E
authors.initials: AE
authors.lastname: Palmer
mesh_terms: D021581:Active Transport, Cell Nucleus; D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D002467:Cell Nucleus / Q000378:metabolism; D003116:Color; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D013997:Time Factors; D015032:Zinc / Q000378:metabolism*
chemical_list: D049452:Green Fluorescent Proteins; D015032:Zinc