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+.

Zinc and diabetes

“…Free cytosolic zinc concentrations measured using these FRET-sensors were approximately in the same range as those measured with chemical probes or hybrid sensors, and it is generally agreed now that free zinc is around a few hundreds of picomolar [53][54][55]. Both FRET sensor families have been targeted to intracellular organelles such as the ER, the nucleus, the mitochondria or the Golgi apparatus by in-frame fusion with suitable targeting motifs [51,[56][57][58]. Of note, concentrations measured in the ER and in the mitochondria varied greatly depending on the FRET or hybrid sensors used, discrepancies that still have to be explained [51,[55][56][57].…”

Genetically-encoded FRET-based sensors for monitoring Zn2+ in living cells

“…Red-shifted variants have also been developed for the Zapbased FRET sensors. 22 The original CFP and YFP domains in ZapCY1 and ZapCY2 were replaced by a variety of red-shifted fluorescent domains. Their performance was assessed in both the cytosol and nucleus of HeLa cells, by in situ calibrations with TPEN and excess Zn 2+ .…”

Intracellular zinc in insulin secretion and action: a determinant of diabetes risk?

“…Shortening of the linker length between the metal binding domains and/or mutation of one of the metal binding cysteines in the WD4 domain yielded a series of sensor variants showing affinities (pM-nM) (7) . Taking advantage of simple fusion with a targeting sequence, several zinc sensors have been addressed to different organelles such as the mitochondria (mitoeCALWY-4, mito-ZapCY1) (8,31) , the endoplasmic reticulum (ER-eCALWY-4, ER-ZapCY1) (8,23) , Golgi apparatus (golgi-ZapCY1) (30) nucleus (NLS-Zaps) (32) and insulinsecreting vesicles (vamp2-eCALWYs, vamp2-eZinCh1) (7) . A summary for the results obtained with these probes is presented in Fig.…”

“…The reasons for these variations are unclear and may involve differences in intracellular pH on which the probes are steeply dependent. Red-shifted variant have been created for Zap and eCALWY sensors (32,34) . Hybrid probes include both genetically-encoded and small molecular elements.…”