Live-Cell Imaging and Analysis with Multiple Genetically Encoded Reporters.

Area of Science:

• Cell Biology
• Systems Biology
• Biophysics

Background:

• Cell signaling pathways exhibit significant cell-to-cell heterogeneity.
• Understanding this heterogeneity is crucial for deciphering cellular functions and responses.
• Live-cell reporters provide high temporal resolution for studying dynamic cellular processes.

Purpose of the Study:

• To present a methodology for designing and conducting live-cell multiplexed reporter experiments.
• To detail methods for analyzing time-course data from single-cell reporter assays.
• To enable quantification of correlations and trends in signaling pathway activity.

Main Methods:

• Utilizing genetically encoded live-cell reporters for signaling pathway activity measurement.
• Employing multiple spectrally distinct reporters within individual cells.
• Analyzing time-course data to quantify single-cell parameters.

Main Results:

• Demonstrated a framework for live-cell multiplexed reporter experiments.
• Established approaches for quantifying signal transmission efficiency and delay between pathway nodes.
• Enabled quantification of correlations between different signaling pathways within single cells.

Conclusions:

• Live-cell multiplexed reporters are powerful tools for dissecting cell signaling heterogeneity.
• This approach facilitates the establishment of mechanisms and consequences of cellular heterogeneity.
• The findings can inform the development of new models for signaling pathway function.