Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology

Abstract

SummaryZinc plays a central role in all living cells as a cofactor for enzymes and as a structural element enabling the adequate folding of proteins. In eukaryotic cells, metals are highly compartmentalized and chelated. Although essential to characterize the mechanisms of Zn 2+ homeostasis, the measurement of free metal concentrations in living cells has proved challenging and the dynamics are difficult to determine.Our ] in response to external supply suggests the involvement of high-and low-affinity uptake systems as well as release from internal stores.In this study, we demonstrate that the combination of genetically encoded FRET sensors and microfluidics provides an attractive tool to monitor the dynamics of cellular metal ion concentrations over a wide concentration range in root cells.

Got to hide your Zn away: Molecular control of Zn accumulation and biotechnological applications

“…The new technology of genetically encoded fluorescent Zn sensor proteins using Förster resonance energy transfer (FRET) enabled the first experimental estimation of free cytosolic [Zn 2+ ] in plant cells. In Zn sufficient conditions, there is about 0.4 nM of cytosolic free [Zn 2+ ] in root cells of Arabidopsis thaliana seedlings [9]. The value is similar to those calculated for animal cells using the same technique [10], but much higher than estimates for prokaryotes [11].…”

“…A promoter cis-element named ZDRE (zinc deficiency response element) is the binding site of AtbZIP19 and AtbZIP23. A set of genes is downregulated in the double mutant, including AtZIP1, 3,4,5,9,12, and AtIRT3, all of which contain ZDRE sequences in their promoters [8].…”

“…X-ray absorption spectroscopy techniques, such as X-ray absorption near-edge spectroscopy (XANES) and X-ray absorption fine structure (EXAFS), allow the determination of speciation and coordination chemistry of specific elements [185], largely improving the resolution in studies on Zn distribution, storage and chelation. The recent use of genome-encoded FRET (Förster resonance energy transfer) sensors to quantify Zn concentrations in plants has opened the possibility to dynamically analyze Zn fluxes with subcellular resolution, in a quantitative manner [9].…”

A simple microfluidic device for live cell imaging ofArabidopsiscotyledons, leaves, and seedlings

“…This system, called the RootChip, is based on a custom-built microfluidic device that channels the growing primary root of an Arabidopsis seedling into a microfluidic channel fabricated on the surface of a microscope cover glass [1,15,16]. The microfluidic channels allow one to flow pulses of chemical treatments across the root tissue while the sample is mounted on a microscope for imaging.…”

Techniques for the analysis of protein-protein interactions in vivo

“…One reason for the popularity of FRET is that, in addition to its use for detecting PPIs, it also can be used with genetically encoded, unimolecular sensors, and it can be used in native species in vivo (Frommer et al, 2009;Uslu and Grossmann, 2016). A range of sensor molecules have been developed or optimized for use in plants, enabling the detection of calcium (Krebs et al, 2012;Wagner et al, 2015), phosphate (Mukherjee et al, 2015), zinc (Lanquar et al, 2014), and abscisic acid (Jones et al, 2014a) as well as physiochemical states of the cell such as pH (Michard et al, 2008) and membrane voltage . One great advantage of using intramolecular FRET sensors lies in the theoretically equal stoichiometry, rendering ratiometric readouts independent of sensor concentration.…”