Genetically Encoded Sensors to Elucidate Spatial Distribution of Cellular Zinc

Abstract

Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn 2؉ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn 2؉ , these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn 2؉ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn 2؉ is taken up into mitochondria following glutamate/Zn 2؉ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn 2؉ signals under different cellular conditions.Although mammalian cells are known to concentrate transition metals, it is now well established that under resting conditions, "free" (e.g. unbound) metals are maintained at extremely low levels. Estimates of the total Zn 2ϩ concentration in mammalian cells typically range from 100 to 500 M (1); yet free Zn 2ϩ concentrations are tightly buffered by proteins such as metallothionein to maintain cytosolic Zn 2ϩ concentrations in the picomolar to nanomolar range (2-5). However, there is emerging evidence that this static picture is dramatically altered by different cellular conditions, such as redox perturbations caused by oxidative stress (6, 7) and cellular signals such as nitric oxide (8 as mitochondrial function (7, 9, 10). Elucidation of the sources and dynamics of these Zn 2ϩ signals would greatly advance our understanding of the interplay between metal regulation and cellular function.There has been a huge effort in the past few years to develop sensitive and selective fluorescent probes to monitor Zn 2ϩ in biological systems. The majority of this work has focused on the generation of small molecule fluorescent indicators (reviewed by Que et al. (11)). Yet there are also examples of sensors based partially on Zn 2ϩ -binding proteins, such as carbonic anhydrase (12) and metallothionein (13), and peptide scaffolds (14). Although many of these sensors have begun to provide insight into Zn 2ϩ concentrations within cells, one limitation is that it is challenging to explicitly target them to subdomains within the cell. Localized probes are necessary to generate a complete picture of cellular Zn 2ϩ homeostasis in mammalian cells. For this reason, sensors that are genetically encode...

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

“…With the growing recognition that labile Zn 2þ plays important roles in influencing signaling pathways and cellular functions, there has been extensive effort over the past few years to develop fluorescent sensors for monitoring Zn 2þ fluxes in cells (5,15,32,33). Previously we developed two zinc sensors that utilized a single zinc finger from Zif268 as the sensing module (ZifCY1…”

“…These transporters help mediate zinc flux into and out of the cell and intracellular organelles. It is well established that Zn 2þ can be concentrated into secretory vesicles (14), and recent studies have demonstrated a labile pool of Zn 2þ in mitochondria (9,15,16), yet little is known about other intracellular organelles such as the endoplasmic reticulum (ER) and Golgi. Numerous proteins found within the secretory pathway require Zn 2þ for their function, including resident ER chaperones such as calnexin and calreticulin, as well as secreted proteins such as metalloproteases and alkaline phosphatases (2).…”

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

“…Two families have been designed by the Palmer group based on zinc fingers: Zif-and Zap-sensors. 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) .…”

Photobiocatalysis: The Power of Combining Photocatalysis and Enzymes

“…Basically the two natural cofactors that act as ubiquitous reducing agents in natural biochemical routes are NADPH and FADH 2 . [8,9] While these two natural mediators act as cofactors in many enzymes, the problem arises from their poor quenching ability to accept electrons from the conduction band of semiconductors, particularly from TiO 2 . To solve this problem, an electron relay acting as mediator of electrons from the photocatalyst particle to the cofactor has to be present as an additional component in the system.…”