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Updated: Jun 21, 2026
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Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy
Published on: October 20, 2014
Integrating Lateral Super-Resolution and Axial Progression Reveals Distinct Clathrin Pit Formation Pathways
Cristopher Thompson1, Aritra Mondal2, Gregory Lafyatis1
1Department of Physics, The Ohio State University, Columbus, Ohio, USA.
Traffic (Copenhagen, Denmark)
|June 20, 2026
Summary
We developed variable-angle total internal reflection fluorescence structured illumination microscopy (vaTIRF-SIM) to visualize clathrin coat organization and movement during live-cell endocytosis. This new method reveals how clathrin coats grow and move in 3D, advancing our understanding of vesicle formation.
Area of Science:
- Cell Biology
- Biophysics
- Microscopy
Background:
- Clathrin-mediated endocytosis (CME) is crucial for vesicle formation at the plasma membrane.
- Existing live-cell imaging methods struggle to link clathrin coat nanoscale organization with 3D dynamics.
- Static structural methods and lower-resolution live-cell techniques limit real-time understanding of CME progression.
Purpose of the Study:
- To develop a live-cell imaging technique combining super-resolution lateral organization with axial sensitivity.
- To visualize the real-time, three-dimensional progression of clathrin coats during CME.
- To investigate the relationship between clathrin coat nanoscale structure and its dynamic movement.
Main Methods:
- Introduction of variable-angle total internal reflection fluorescence structured illumination microscopy (vaTIRF-SIM).
- Integration of TIRF-SIM with controlled variation of evanescent field penetration depth.
- Application to genome-edited SUM-159 cells expressing AP2-EGFP.
Main Results:
- vaTIRF-SIM enables simultaneous visualization of clathrin coat architecture and axial displacement with high spatiotemporal resolution.
- Coordinated lateral growth and progressive axial advancement of de novo clathrin-coated pits were observed.
- Two distinct endocytic behaviors at clathrin plaques were identified: peripheral and rapid subdomain internalization.
Conclusions:
- vaTIRF-SIM provides the first real-time coupling of nanoscale clathrin coat organization with axial progression during CME.
- The findings reveal dynamic curvature generation and distinct endocytic pathways associated with clathrin plaques.
- This technique offers new insights into the mechanics of vesicle formation in living cells.
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