Techniques for measuring cellular zinc

Abstract

The development and improvement of fluorescent Zn²⁺ sensors and Zn²⁺ imaging techniques have increased our insight into this biologically important ion. Application of these tools has identified an intracellular labile Zn²⁺ pool and cultivated further interest in defining the distribution and dynamics of labile Zn²⁺. The study of Zn²⁺ in live cells in real time using sensors is a powerful way to answer complex biological questions. In this review, we highlight newly engineered Zn²⁺ sensors, methods to test whether the sensors are accessing labile Zn²⁺, and recent studies that point to the challenges of using such sensors. Elemental mapping techniques can complement and strengthen data collected with sensors. Both mass spectrometry-based and X-ray fluorescence-based techniques yield highly specific, sensitive, and spatially resolved snapshots of metal distribution in cells. The study of Zn²⁺ has already led to new insight into all phases of life from fertilization of the egg to life-threatening cancers. In order to continue building new knowledge about Zn²⁺ biology it remains important to critically assess the available toolset for this endeavor.

Keywords: Cells; Elemental analysis; Fluorescent sensors; Genetically encoded sensors; Imaging; Zinc.

Figure 1

Panel A) Comparison of properties of small molecule and FRET based protein sensors. Panel B) Small molecule probes typically contain a fluorophore attached to a Zn²⁺ chelating moiety (FluoZin-3 is shown as an example). FRET-based protein probes typically contain two fluorescent proteins separated by a Zn²⁺-binding domain. Small molecule probes tend to increase in fluorescence intensity upon binding Zn²⁺, whereas FRET-based probes are characterized by an increase in intensity at one wavelength and decrease at another (Sample fluorescence emission spectra are depicted with arrows indicating the change upon Zn²⁺ addition.) Panel C) Most commonly used probes and sensors. Companies and labs that provide these sensors for general use are included in parentheses.

Figure 2

Diagrams of Zn²⁺ calibrations: Panel A. depicts a sensor that increases in FRET ratio when Zn²⁺ binds. Sensors with this mechanism include the Zif, Zap, and eZinCh families. Panel B. depicts a sensor that decreases in FRET ratio when Zn²⁺ binds. The eCALWY family reports Zn²⁺ abundance by this mechanism. The equations used to determine the resting concentration of zinc are given where n equals the hill coefficient and K_D is the dissociation constant determined by titration of the sensor in vitro [–31]. These equations assume that the probes form a 1:1 complex, that the probes are at a low enough concentration in cells for the fluorescence intensity to be linearly proportional to the concentration, and that the probes behave similarly in cells and in vitro [22].

Figure 3

Schematic of mass-spectrometry and X-ray fluorescence-based imaging techniques. A) Mass-spectrometry-based imaging techniques rely on point-by-point sample ablation using a laser or primary ion beam to dispel charged particles or secondary ions that are subsequently analyzed by mass-spectrometry. Ions are separated by their mass-to-charge ratio. B) X-ray fluorescence techniques rely on the photoelectric effect; when an incident beam (X-ray or e⁻) bombards a sample, electrons are ejected from inner shells and vacancies are filled by outer-shell electrons. In this process, X-rays are released and detected at characteristic energies for each element.