Measuring steady-state and dynamic endoplasmic reticulum and Golgi Zn ²⁺ with genetically encoded sensors

Abstract

Zn 2+ plays essential roles in biology, and cells have adopted exquisite mechanisms for regulating steady-state Zn 2+ levels. Although much is known about total Zn 2+ in cells, very little is known about its subcellular distribution. Yet defining the location of Zn 2+ and how it changes with signaling events is essential for elucidating how cells regulate this essential ion. Here we create fluorescent sensors genetically targeted to the endoplasmic reticulum (ER) and Golgi to monitor steady-state Zn 2+ levels as well as flux of Zn 2+ into and out of these organelles. These studies reveal that ER and Golgi contain a concentration of free Zn 2+ that is 100 times lower than the cytosol. Both organelles take up Zn 2+ when cytosolic levels are elevated, suggesting that the ER and Golgi can sequester elevated cytosolic Zn 2+ and thus have the potential to play a role in influencing Zn 2+ toxicity. ER Zn 2+ homeostasis is perturbed by small molecule antagonists of Ca 2+ homeostasis and ER Zn 2+ is released upon elevation of cytosolic Ca 2+ pointing to potential exchange of these two ions across the ER. This study provides direct evidence that Ca 2+ signaling can influence Zn 2+ homeostasis and vice versa, that Zn 2+ dynamics may modulate Ca 2+ signaling.

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].…”

“…in the low nanomolar range) has recently allowed the measurement of free Zn 2+ concentration in living cells with high precision and spatial accuracy. Two main families of FRET-based Zn 2+ sensors have been developed, namely the Zap-sensors by the Palmer group [51], and the eCALWYsensors by the Merkx group and ourselves [52]. 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].…”

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

“…The low (μM) affinity Zif family is derived from the mammalian transcription factor Zif268, and contains either a wild type zinc fingers (ZifCY1), or a mutated (ZifCY2) domain (29,30) . The Zap sensors, based on the Saccharomyces cerevisiae transcriptional regulator Zap1, have a very high (pM) affinity for zinc (30) . The first member of the family ZapCY1 showed a K d of 2·5 pM, and was saturated when expressed in HeLa cells.…”

“…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.…”