Quantitative Mapping of Nanoscale EGFR-Grb2 Assemblies by DNA-PAINT

Area of Science:

• Cellular Biology
• Molecular Biology
• Biophysics

Background:

• Receptor tyrosine kinase (RTK) signaling is crucial for cellular functions.
• Understanding the nanoscale organization of membrane protein assemblies is vital for signal transduction.
• Existing methods struggle to resolve the heterogeneity and small size of these assemblies.

Purpose of the Study:

• To develop and validate a single-molecule super-resolution imaging and analysis workflow.
• To quantitatively characterize individual membrane protein assembly sites in situ.
• To investigate the nanoscale organization of epidermal growth factor receptor (EGFR) and Grb2.

Main Methods:

• Developed a novel single-molecule super-resolution imaging workflow.
• Applied computational analysis to resolve nanoscale protein organization.
• Studied EGFR and Grb2 dynamics in response to epidermal growth factor stimulation.

Main Results:

• Observed a decrease in EGFR density upon stimulation.
• Quantified the accumulation of Grb2 at EGFR assembly sites.
• Detected an increase in both dimeric and higher-order EGFR oligomers during activation.

Conclusions:

• The new workflow enables quantitative analysis of membrane protein assemblies at the single-molecule level.
• Revealed dynamic nanoscale organization changes of EGFR and Grb2 during signaling.
• The framework is broadly applicable to studying various membrane protein assemblies.