Ulation of cellular Zn2+ levels. The sensors were then targeted to both the nucleus and cytosol and nuclear sensors were used in conjunction with an organelle-localized CFPYFP-based Zn2+ sensor to monitor Zn2+ fluxes in two cellular compartments simultaneously. We believe these represent an important breakthrough in expanding the palette of Zn2+ sensors.Cell Culture and MicroscopyHeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies) supplemented with 10 (v/v) fetal bovine serum (Atlanta Biologicals), 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were incubated at 37uC in 5 CO2, changing the media every 3 days. Once cells were approximately 80?0 confluent they were split and seeded onto 3.5 cm imaging dishes until they were approximately 40?0 confluent. At this point 1 mg of sensor DNA was transiently transfected using TransITH-LT1 (Mirus) as specified by manufacturer instructions. Forty-eight hours after transfection, cells were imaged using phosphate, calcium, and magnesium free HEPES-buffered Hanks’Methods FRET Sensor CloningA schematic of the general sensor construction is presented in 58-49-1 chemical information Figure 1A and all sequences have been MedChemExpress SPI1005 deposited in GenBank. Table S1 summarizes the mutations in the zinc finger domain. For bacterial expression, sensor cDNA was cloned into pET302/ NT-His (Life Technologies) in which the BamHI and EcoRI restriction sites were reversed. For mammalian cell expression, Table 1. Fluorescent Protein Excitation and Emission.Donor FP CFP tSapphire tSapphire mOrange2 mOrange2 Clover Clover CloverAcceptor FP YFP mKO TagRFP mCherry mKATE mRuby2 mRuby2 
mRubySensor Name ZapCY2 ZapSM2 ZapSR2 ZapOC2 ZapOK2 ZapCmR1 ZapCmR1.1 ZapCmRExcitation max (nm) 435 399 399 549 549 486 486Emission max (nm) 535 559 580 610 633 605 605Senor nomenclature is as follows: Zap refers to the 1st two zinc fingers of the Saccaromyces cerevisiae Zap1 transcription factor that serves as the zinc binding domain, the next two letters refer to the donor and acceptor FPs, finally the “1” at the end of a sensor name indicates the wild type Zap1 domain was used, the “2” indicates that two mutations (Cys to His) were incorporated to lower the zinc affinity, as outlined in [15]. “1.1” indicates one mutation (Cys to His) was incorporated. doi:10.1371/journal.pone.0049371.tAlternately Colored FRET Sensors for ZincAlternately Colored FRET Sensors for ZincFigure 2. FRET Sensor calibration in the nucleus. Representative calibrations of each sensor localized to the nucleus. The background corrected FRET ratio (FRET Intensity 4 Donor Intensity) is represented as a function of time. Calibrations were performed by adding 150 mM TPEN to achieve RTPEN, followed by washing of residual TPEN and addition of 135 mM ZnCl2 with 10 mM Digitonin to permeabilize the cell membrane and obtain RZn. A) NLS-ZapSM2 FRET ratio increases slightly above resting suggesting that it is close to saturation at rest; B) NLS-ZapSR2, FRET ratio goes above resting; C) NLS-ZapOC2 has a small decrease in FRET 
ratio after TPEN and a larger increase 16574785 after treatment with Zn2+; D) NLS-ZapOK2 exhibits a small change in FRET ratio after TPEN and Zn2+; E) NLS-ZapCmR1 has an inverted response in which TPEN causes an increase in FRET ratio while Zn2+ with digitonin causes a decrease in the ratio; F) NLS-ZapCmR1.1 displays a decrease in the FRET ratio after TPEN and large increase with Zn2+ and digitonin; G) NLS-ZapCmR2 is similar to ZapCmR1.1. Representative t.Ulation of cellular Zn2+ levels. The sensors were then targeted to both the nucleus and cytosol and nuclear sensors were used in conjunction with an organelle-localized CFPYFP-based Zn2+ sensor to monitor Zn2+ fluxes in two cellular compartments simultaneously. We believe these represent an important breakthrough in expanding the palette of Zn2+ sensors.Cell Culture and MicroscopyHeLa cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies) supplemented with 10 (v/v) fetal bovine serum (Atlanta Biologicals), 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were incubated at 37uC in 5 CO2, changing the media every 3 days. Once cells were approximately 80?0 confluent they were split and seeded onto 3.5 cm imaging dishes until they were approximately 40?0 confluent. At this point 1 mg of sensor DNA was transiently transfected using TransITH-LT1 (Mirus) as specified by manufacturer instructions. Forty-eight hours after transfection, cells were imaged using phosphate, calcium, and magnesium free HEPES-buffered Hanks’Methods FRET Sensor CloningA schematic of the general sensor construction is presented in Figure 1A and all sequences have been deposited in GenBank. Table S1 summarizes the mutations in the zinc finger domain. For bacterial expression, sensor cDNA was cloned into pET302/ NT-His (Life Technologies) in which the BamHI and EcoRI restriction sites were reversed. For mammalian cell expression, Table 1. Fluorescent Protein Excitation and Emission.Donor FP CFP tSapphire tSapphire mOrange2 mOrange2 Clover Clover CloverAcceptor FP YFP mKO TagRFP mCherry mKATE mRuby2 mRuby2 mRubySensor Name ZapCY2 ZapSM2 ZapSR2 ZapOC2 ZapOK2 ZapCmR1 ZapCmR1.1 ZapCmRExcitation max (nm) 435 399 399 549 549 486 486Emission max (nm) 535 559 580 610 633 605 605Senor nomenclature is as follows: Zap refers to the 1st two zinc fingers of the Saccaromyces cerevisiae Zap1 transcription factor that serves as the zinc binding domain, the next two letters refer to the donor and acceptor FPs, finally the “1” at the end of a sensor name indicates the wild type Zap1 domain was used, the “2” indicates that two mutations (Cys to His) were incorporated to lower the zinc affinity, as outlined in [15]. “1.1” indicates one mutation (Cys to His) was incorporated. doi:10.1371/journal.pone.0049371.tAlternately Colored FRET Sensors for ZincAlternately Colored FRET Sensors for ZincFigure 2. FRET Sensor calibration in the nucleus. Representative calibrations of each sensor localized to the nucleus. The background corrected FRET ratio (FRET Intensity 4 Donor Intensity) is represented as a function of time. Calibrations were performed by adding 150 mM TPEN to achieve RTPEN, followed by washing of residual TPEN and addition of 135 mM ZnCl2 with 10 mM Digitonin to permeabilize the cell membrane and obtain RZn. A) NLS-ZapSM2 FRET ratio increases slightly above resting suggesting that it is close to saturation at rest; B) NLS-ZapSR2, FRET ratio goes above resting; C) NLS-ZapOC2 has a small decrease in FRET ratio after TPEN and a larger increase 16574785 after treatment with Zn2+; D) NLS-ZapOK2 exhibits a small change in FRET ratio after TPEN and Zn2+; E) NLS-ZapCmR1 has an inverted response in which TPEN causes an increase in FRET ratio while Zn2+ with digitonin causes a decrease in the ratio; F) NLS-ZapCmR1.1 displays a decrease in the FRET ratio after TPEN and large increase with Zn2+ and digitonin; G) NLS-ZapCmR2 is similar to ZapCmR1.1. Representative t.