Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Tuesday Speaker Abstracts

Systematic Evaluation of Cellular Zn 2+ Sensors with Microfluidic Cytometry Brett Fiedler 1,2 , Kyle P. Carter 2,3 , Yan Qin 4 , Margaret C. Carpenter 2,3 , Amy E. Palmer 2,3 , Ralph Jimenez 1,2 . 1 University of Colorado, Boulder, CO, USA, 2 University of Colorado, Boulder, CO, USA, 3 University of Colorado, Boulder, CO, USA, 4 University of Denver, Denver, CO, USA. The accuracy of cellular ion concentration measurements with genetically-encoded biosensors depends heavily on heterogeneity of biosensor expression and on the reproducibility of the response as a function of environment. Studies with currently-available Zn 2+ FRET-biosensors have reported widely varying Zn 2+ concentrations in the endoplasmic reticulum (ER). It was previously observed that these sensors show a smaller dynamic range of FRET change in the ER relative to the cytosol. Furthermore, it has been observed that sensors with small dynamic ranges do not accurately report analyte concentrations. To investigate this discrepancy, we employed microfluidic flow cytometry to analyze the heterogeneity of the apo state of three Zn 2+ biosensors (ZapCY1, eCALWY-4, eZinCh-2) localized to the cytosol and to the ER of HeLa cells. These sensors utilize three distinctly different moieties for Zn 2+ binding. This instrument was also used to probe the responses of the sensors to perturbations of cellular Zn 2+ concentrations on the timescales of several seconds. For each sensor, we find the absolute FRET value of the apo state shows an offset and larger heterogeneity in the ER relative to the cytosol. Secondly, the screening of the sensor dynamic responses revealed multiple subpopulations. Third, we found that sensor performance in the cytosol is not always correlated with high performance in other cellular compartments. Finally, we are using this microfluidic system to increase dynamic range of ER-localized FRET sensors by screening and sorting a cell- based library that targets the linker regions between the donor/acceptor FPs and Zn 2+ binding domains.

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