  We present a single-molecule tool called the CoPro (Concentration of
Proteins) method that uses millisecond imaging with convolution analysis,
automated image segmentation and super-resolution localization microscopy to
generate robust estimates for protein concentration in different compartments
of single living cells, validated using realistic simulations of complex
multiple compartment cell types. We demonstrates its utility experimentally on
model Escherichia coli bacteria and Saccharomyces cerevisiae budding yeast
cells, and use it to address the biological question of how signals are
transduced in cells. Cells in all domains of life dynamically sense their
environment through signal transduction mechanisms, many involving gene
regulation. The glucose sensing mechanism of S. cerevisiae is a model system
for studying gene regulatory signal transduction. It uses the multi-copy
expression inhibitor of the GAL gene family, Mig1, to repress unwanted genes in
the presence of elevated extracellular glucose concentrations. We fluorescently
labelled Mig1 molecules with green fluorescent protein (GFP) via chromosomal
integration at physiological expression levels in living S. cerevisiae cells,
in addition to the RNA polymerase protein Nrd1 with the fluorescent protein
reporter mCherry. Using CoPro we make quantitative estimates of Mig1 and Nrd1
protein concentrations in the cytoplasm and nucleus compartments on a
cell-by-cell basis under physiological conditions. These estimates indicate a
4-fold shift towards higher values in concentration of diffusive Mig1 in the
nucleus if the external glucose concentration is raised, whereas equivalent
levels in the cytoplasm shift to smaller values with a relative change an order
of magnitude smaller. This compares with Nrd1 which is not involved directly in
glucose sensing, which is almost exclusively localized in the nucleus under
high and...
