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. 2020 Feb 26;9:e53638. doi: 10.7554/eLife.53638

Figure 2. Sens Fano factor relative to protein copy number in single cells.

(A) sfGFP- and mCherry-Sens protein numbers measured from single cells in sfGFP-sens/mCherry-sens wing discs. 12,000 cells were plotted for comparison in (A,B). Colors represent cell count in each hexagonal plot region. (B) Estimated sfGFP and mCherry numbers generated from the tandem-tagged sfGFP-mCherry-sens gene in single cells of wing discs. Note that these numbers are not perfectly correlated with one another due to noise in the measurement process (see also Figure 2—figure supplement 1). As expected, median expression of the tandem tag was 2.1 ± 0.03 fold higher than allelic tag expression. (C) Top panel: The Fano factor was calculated in bins of cells expressing either tandem-tagged Sens or the singly-tagged allelic pairs of Sens (Figure 2—figure supplement 2). Bottom panel: The Fano factor of Sens expression was calculated by subtracting out the Fano factor from tandem-tagged cells. Plotted region encompasses data from 88% of tagged cells (Sens < 1000 molecules). Shading demarcates 95% confidence intervals.

Figure 2.

Figure 2—figure supplement 1. Fluorescence Resonance Energy Transfer (FRET) from sfGFP to mCherry molecules is negligible under experimental imaging conditions.

Figure 2—figure supplement 1.

Wing disc cells with tandem tagged sfGFP-mCherry-sens alleles were imaged under identical conditions with either both green and red lasers ‘on’ to assay stochastic noise, or with only the green laser ‘on’ to assay FRET from sfGFP to mCherry molecules. Raw single cell green channel fluorescence intensity is plotted on the x-axis. All cells in the imaging field were included for analysis. Red channel fluorescence values, on y-axis, were linearly transformed to make the slope equal to 1 and y-intercept equal to 0 for noise assay data. Single cell red fluorescence values for FRET assay data were then transformed with identical parameters for comparison.
Figure 2—figure supplement 2. Fano factor calculation from nuclear fluorescence signals of Sens positive cells.

Figure 2—figure supplement 2.

(A) The mCherry signal intensity is scaled relative to sfGFP signal in Sens-positive cells from individual discs. (B) Data from discs of the same genotype are pooled together. (C) Pooled data are sorted into separate bins according to total Sens output. The intrinsic noise and mean for Sens is calculated for each binned sub-population. (D) The Fano factor of each bin is plotted as a function of mean Sens output in a bin. 95% confidence intervals for the estimated Fano factor are calculated by bootstrapping with resampling within each binned sub- population.