Mapping Epidermal Growth Factor Receptor-1 Sorting Domains in Endosomes with a Calibrated Three-Dimensional Expansion...

Area of Science:

• Cell Biology
• Microscopy
• Nanotechnology

Background:

• Endosomes are crucial for sorting and recycling cell-surface receptors like EGFR1.
• Nanoscale interactions within endosomes are vital but difficult to visualize with current microscopy.
• Previous methods struggled with accurate 3D visualization of small intracellular compartments.

Purpose of the Study:

• To adapt and validate expansion microscopy (ExM) for visualizing and quantifying endosomal protein nanoclusters.
• To develop a method for correcting expansion distortions in ExM images of endosomes.
• To provide a quantitative framework for studying molecular-scale events in endosome dynamics.

Main Methods:

• Adapted expansion microscopy (ExM) for 3D visualization of endosomal proteins in RPE-1 cells.
• Developed 3D distortion analysis using optical-flow principles to correct for hydrogel expansion anisotropies.
• Introduced a protein nanocage for calibrating local nanoscale expansion factors.
• Applied pulse-chase labeling of EGF/EGFR1 and multiplexed 3D Airyscan microscopy.
• Utilized a volume tracing pipeline to analyze protein density changes.

Main Results:

• Identified under-expansion in cytoplasmic regions and overestimation of endosome size/distance in standard ExM.
• Successfully calibrated ExM images using a protein nanocage reporter.
• Visualized EGF and EGFR1 internalization and sorting dynamics.
• Observed enrichment of EGF/EGFR1 in endosomal interiors and Rab5a accumulation during early endosome maturation.

Conclusions:

• The developed multiplexed 3D ExM toolkit enables accurate visualization and measurement of nanoscale endosomal structures.
• This quantitative framework overcomes limitations of conventional and super-resolution microscopy for small organelles.
• Provides new insights into the molecular mechanisms of EGFR1 sorting and endosome maturation.