Phenotyping by Molecular Mobility: Low-Affinity Reversible Probes Enable Characterizing and Classifying Cells...

Area of Science:

• Cell Biology
• Biophysics
• Molecular Imaging

Background:

• Cell surface receptors are crucial for cellular functions and vary across cell types.
• Understanding receptor behavior is key to differentiating cell functions and pathogenicity.
• Existing methods often require genetic modification or labeling, perturbing native cell states.

Purpose of the Study:

• To present a super-resolution method for functional cell phenotyping using single-molecule analysis of membrane receptor mobility.
• To enable the study of endogenous receptors in unperturbed live cells.
• To establish a workflow for cell phenotyping based on receptor mobility patterns.

Main Methods:

• Development of nanofitin-based low-affinity probes for selective, reversible binding to target receptors.
• Single-molecule tracking and analysis of membrane receptor mobility in live cells.
• Integration with super-resolution microscopy (Point Accumulation for Imaging in Nanoscale Topography) and AI-driven classification.

Main Results:

• Demonstrated that identical receptors exhibit distinct mobility patterns across different cell types.
• Showcased the ability to detect, count, and track individual endogenous receptors without genetic modification or labeling.
• Validated the method for functional phenotyping of cell populations based on receptor mobility.

Conclusions:

• The presented super-resolution method provides a powerful tool for functional cell phenotyping.
• Analysis of membrane receptor mobility offers insights into underlying physiological differences between cell types.
• This approach allows for the study of endogenous biomolecules in their native cellular environment.