Monstrating these compartments play an important role in cytosolic clearance and maintenance of homeostasis [15,36]. However for these prior experiments accumulation of Zn2+ in the cytosol and intracellular organelles was measured in separate experiments making it impossible to directly compare the relative rate of uptake in individual cells. To observe Zn2+ uptake into the nucleus we used either NLS-ZapSR2 or NLS-ZapCmR2 and ZapCY1 was targeted to individual organelles. Figure 5a depicts representative images showing sensor localization. As observed in Figure 5d , addition of 100 mM ZnCl2 to the extracellular milieu, gave rise to an immediate MedChemExpress Ornipressin increase in nuclear Zn2+, whereas organelle Zn2+ (ER, Golgi, and mitochondria, Figure 5d, 5e, and 5f) increases approximately 600?00 seconds later.Alternately Colored FRET Sensors for ZincFigure 5. Simultaneous order AZ 876 monitoring of Zn2+ uptake into the nucleus and either the endoplasmic reticulum, Golgi apparatus, or mitochondria. Representative images (FRET channel) and FRET ratio traces of Zn2+ uptake into the nucleus, ER, Golgi, or mitochondria. A) Image of nuclear and ER FRET sensor, left panel illustrates NLS-ZapSR2, middle panel ER-ZapCY1 and right panel is a pseudo-color merged image of NLSZapSR2 and ER-ZapCY1. B) Image of nuclear and ER FRET sensor, left panel illustrates NLS-ZapSR2, middle panel Golgi-ZapCY1 and right panel is a pseudo- color merged image of NLS-ZapSR2 and Golgi-ZapCY1. C) Image of nuclear and mitochondrial FRET sensor, left panel illustrates NLSZapCmR2, middle panel mitochondria-ZapCY1 and right panel is a pseudo- color merged image of NLS-ZapCmR2 and mitochondria-ZapCY1. D-F) FRET ratio traces of NLS-ZapSR2 or NLS-ZapCmR2 with ER-, Golgi-, and mitochondrial-ZapCY1 upon addition of 100 mM extracellular ZnCl2 at the time indicated. The nuclear FRET ratio rises more rapidly than organelle FRET ratio. The organelle FRET ratio begins to increase approximately 600 seconds post-Zn2+. Experiments were repeated at least five times with a minimum of 1? cells per experiment. All images were bleedthrough corrected. Scale bar = 20 mm. doi:10.1371/journal.pone.0049371.gConclusionsHere we report the first alternatively colored Zn sensors constructed from green, orange, and red FPs that can be used simultaneously with a CFP/YFP sensor. Given evidence that changes in cellular Zn2+ have been linked with changes in other ions such as Ca2+ [15,37] and signaling pathways such as the MAPK pathway [38] and apoptotic cascades [39,40], a broad palette of Zn2+ sensors that permits simultaneous monitoring of multiple events would be useful tools to provide mechanistic insight into these connections. Sensors were targeted to both the cytosol and nucleus and intriguingly, 6 of the 7 sensors registered a higher fractional saturation in the nucleus compared to the cytosol, suggesting that nuclear Zn2+ may be buffered at a higher concentration than cytosolic Zn2+. Although there are currently no estimates of nuclear Zn2+ levels, given the large number of transcription factors that bind Zn2+ [41], it seems reasonable to speculate the nuclear buffering system may differ from that in the cytosol.2+The development of multi-color FRET sensors for Zn2+ allowed us to monitor Zn2+ simultaneously in the nucleus and other organelles, such as the ER, Golgi, or mitochondria. For these experiments we measured Zn2+ uptake or sequestration following acute elevation of extracellular Zn2+. Extracellular Zn2+ levels are typi.Monstrating these compartments play an important role in cytosolic clearance and maintenance of homeostasis [15,36]. However for these prior experiments accumulation of Zn2+ in the cytosol and intracellular organelles was measured in separate experiments making it impossible to directly compare the relative rate of uptake in individual cells. To observe Zn2+ uptake into the nucleus we used either NLS-ZapSR2 or NLS-ZapCmR2 and ZapCY1 was targeted to individual organelles. Figure 5a depicts representative images showing sensor localization. As observed in Figure 5d , addition of 100 mM ZnCl2 to the extracellular milieu, gave rise to an immediate increase in nuclear Zn2+, whereas organelle Zn2+ (ER, Golgi, and mitochondria, Figure 5d, 5e, and 5f) increases approximately 600?00 seconds later.Alternately Colored FRET Sensors for ZincFigure 5. Simultaneous monitoring of Zn2+ uptake into the nucleus and either the endoplasmic reticulum, Golgi apparatus, or mitochondria. Representative images (FRET channel) and FRET ratio traces of Zn2+ uptake into the nucleus, ER, Golgi, or mitochondria. A) Image of nuclear and ER FRET sensor, left panel illustrates NLS-ZapSR2, middle panel ER-ZapCY1 and right panel is a pseudo-color merged image of NLSZapSR2 and ER-ZapCY1. B) Image of nuclear and ER FRET sensor, left panel illustrates NLS-ZapSR2, middle panel Golgi-ZapCY1 and right panel is a pseudo- color merged image of NLS-ZapSR2 and Golgi-ZapCY1. C) Image of nuclear and mitochondrial FRET sensor, left panel illustrates NLSZapCmR2, middle panel mitochondria-ZapCY1 and right panel is a pseudo- color merged image of NLS-ZapCmR2 and mitochondria-ZapCY1. D-F) FRET ratio traces of NLS-ZapSR2 or NLS-ZapCmR2 with ER-, Golgi-, and mitochondrial-ZapCY1 upon addition of 100 mM extracellular ZnCl2 at the time indicated. The nuclear FRET ratio rises more rapidly than organelle FRET ratio. The organelle FRET ratio begins to increase approximately 600 seconds post-Zn2+. Experiments were repeated at least five times with a minimum of 1? cells per experiment. All images were bleedthrough corrected. Scale bar = 20 mm. doi:10.1371/journal.pone.0049371.gConclusionsHere we report the first alternatively colored Zn sensors constructed from green, orange, and red FPs that can be used simultaneously with a CFP/YFP sensor. Given evidence that changes in cellular Zn2+ have been linked with changes in other ions such as Ca2+ [15,37] and signaling pathways such as the MAPK pathway [38] and apoptotic cascades [39,40], a broad palette of Zn2+ sensors that permits simultaneous monitoring of multiple events would be useful tools to provide mechanistic insight into these connections. Sensors were targeted to both the cytosol and nucleus and intriguingly, 6 of the 7 sensors registered a higher fractional saturation in the nucleus compared to the cytosol, suggesting that nuclear Zn2+ may be buffered at a higher concentration than cytosolic Zn2+. Although there are currently no estimates of nuclear Zn2+ levels, given the large number of transcription factors that bind Zn2+ [41], it seems reasonable to speculate the nuclear buffering system may differ from that in the cytosol.2+The development of multi-color FRET sensors for Zn2+ allowed us to monitor Zn2+ simultaneously in the nucleus and other organelles, such as the ER, Golgi, or mitochondria. For these experiments we measured Zn2+ uptake or sequestration following acute elevation of extracellular Zn2+. Extracellular Zn2+ levels are typi.
