4 years ago

Critical Comparison of FRET-Sensor Functionality in the Cytosol and Endoplasmic Reticulum and Implications for Quantification of Ions

Critical Comparison of FRET-Sensor Functionality in the Cytosol and Endoplasmic Reticulum and Implications for Quantification of Ions
Ralph Jimenez, Margaret C. Carpenter, Kyle P. Carter, Amy E. Palmer, Brett Fiedler
Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for quantifying and visualizing analytes in living cells, and when targeted to organelles have the potential to define distribution of analytes in different parts of the cell. However, quantitative estimates of analyte distribution require rigorous and systematic analysis of sensor functionality in different locations. In this work, we establish methods to critically evaluate sensor performance in different organelles and carry out a side-by-side comparison of three different genetically encoded sensor platforms for quantifying cellular zinc ions (Zn2+). Calibration conditions are optimized for high dynamic range and stable FRET signals. Using a combination of single-cell microscopy and a novel microfluidic platform capable of screening thousands of cells in a few hours, we observe differential performance of these sensors in the cytosol compared to the ER of HeLa cells, and identify the formation of oxidative oligomers of the sensors in the ER. Finally, we use new methodology to re-evaluate the binding parameters of these sensors both in the test tube and in living cells. Ultimately, we demonstrate that sensor responses can be affected by different cellular environments, and provide a framework for evaluating future generations of organelle-targeted sensors.

Publisher URL: http://dx.doi.org/10.1021/acs.analchem.7b02933

DOI: 10.1021/acs.analchem.7b02933

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