Millisecond single-molecule localization microscopy combined with convolution analysis and automated image...

Area of Science:

• Cellular and Molecular Biology
• Biophysics
• Microscopy and Imaging

Background:

• Cells dynamically sense their environment via signal transduction mechanisms, often involving gene regulation.
• The glucose sensing mechanism in Saccharomyces cerevisiae, utilizing the Mig1 repressor, serves as a model for gene regulatory signal transduction.
• Understanding protein concentration dynamics is crucial for deciphering cellular responses to environmental cues.

Purpose of the Study:

• To introduce and validate the CoPro (concentration of proteins) method for quantifying protein concentrations in single living cells.
• To investigate the dynamic changes in protein concentrations within cellular compartments in response to external stimuli, specifically glucose levels.
• To apply the CoPro method to study signal transduction in Saccharomyces cerevisiae, focusing on the Mig1 protein.

Main Methods:

• Development of the CoPro method combining millisecond imaging, convolution analysis, automated image segmentation, and super-resolution localization microscopy.
• Experimental validation using model organisms: Escherichia coli and Saccharomyces cerevisiae.
• Fluorescent labeling of Mig1 and Nrd1 proteins in S. cerevisiae using GFP and mCherry, respectively, at physiological expression levels.

Main Results:

• The CoPro method accurately estimates protein concentrations in different cellular compartments, validated by simulations.
• A ~4-fold increase in nuclear Mig1 concentration was observed in S. cerevisiae upon elevated external glucose, with a smaller decrease in the cytoplasm.
• Nrd1 protein, not directly involved in glucose sensing, showed stable nuclear localization irrespective of glucose levels.

Conclusions:

• The CoPro method provides robust, time-resolved quantification of protein concentrations in single cells and across populations.
• This technique enables the study of subtle and rapid changes in protein concentration crucial for understanding cellular processes and signal transduction.
• CoPro facilitates a deeper understanding of how environmental changes are translated into cellular responses at the molecular level.