44 / 155
Single-Cell Biophysics: Measurement, Modulation, and Modeling
Tuesday Speaker Abstracts
39
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.