Buccella Group Strategy for Sensing Magnesium Ion Concentration, in JACS
|In a JACS paper entitled "Visualizing Compartmentalized Cellular Mg2+ on Demand with Small-Molecule Fluorescent Sensors," the Buccella Group describes a general approach for sensing magnesium ion concentration changes inside specific cellular organelles. Methods for quantification of Mg2+ in cells and tissues are needed to establish the role of this metal cation in sustaining metabolic activities required for cell proliferation and death. In this paper, Professor Daniela Buccella and graduate students Jessica Gruskos and Guangqian Zhang introduce a two-step strategy for in situ anchoring and activation of a fluorescent Mg2+ sensor within an organelle of choice. The group is utilizing this tool to probe changes in Mg2+ distribution as a function of cellular state.|
Abstract: The study of intracellular metal ion compartmentalization and trafficking involved in cellular processes demands sensors with controllable localization for the measurement of organelle-specific levels of cations with subcellular resolution. We introduce herein a new two-step strategy for in situ anchoring and activation of a fluorescent Mg2+ sensor within an organelle of choice, using a fast fluorogenic reaction between a tetrazine-functionalized pro-sensor, Mag-S-Tz, and a strained bicyclononyne conjugated to a genetically encoded HaloTag fusion protein of known cellular localization. Protein conjugation does not affect the metal-binding properties of the o-aminophenol-N,N,O-triacetic acid (APTRA)-based fluorescent indicator, which displays a dissociation constant Kd = 3.1 mM suitable for the detection of low millimolar concentrations of chelatable Mg2+ typical of the intracellular environment. We demonstrate the application of our sensing system for the ratiometric detection of Mg2+ in target organelles in HEK 293T cells, providing the first direct comparison of subcellular pools of the metal without interfering signal from other compartments. Activation of the fluorescence in situ through a fluorogenic conjugation step effectively constrains the fluorescence signal to the locale of interest, thus improving the spatial resolution in imaging applications and eliminating the need for washout of mislocalized sensor. The labeling strategy is fully compatible with live cell imaging, and provides a valuable tool for tracking changes in metal distribution that to date have been an unsolved mystery in magnesium biology.
This research was supported by the National Science Foundation and the National Institutes of Health.
Updated on 01/19/2017