Single-laser polarization FRET (polFRET) on the cell surface

Area of Science:

• Biophysics
• Cell Biology
• Immunology

Background:

• Cell surface receptor interactions are crucial for cellular communication and function.
• Understanding receptor mobility and clustering provides insights into signaling pathways.
• Existing methods may lack the precision to simultaneously measure rotational mobility and proximity.

Purpose of the Study:

• To develop and validate a new method for simultaneous detection of rotational mobility and proximity of cell surface receptors.
• To quantify FRET efficiency, rotational characteristics, and FRET-fraction of receptors on a cell-by-cell basis.
• To apply the method to study MHCI and MHCII receptors on human lymphoblast cells.

Main Methods:

• Utilized a Förster Resonance Energy Transfer (FRET) system with donor and acceptor fluorophores.
• Employed cell-by-cell measurement of polarized fluorescence intensity components.
• Performed flow cytometry and time-resolved fluorescence lifetime imaging microscopy (rFLIM) measurements.
• Measured fluorescence anisotropy of donor and acceptor, and FRET efficiency at varying acceptor-to-donor ratios.

Main Results:

• The developed method successfully determines FRET efficiency, donor/acceptor concentrations, rotational characteristics, and FRET-fraction.
• Acceptor anisotropy was found to be more sensitive than donor anisotropy for FRET detection.
• Successfully determined rotational constants and associated fractions of MHCI and MHCII molecules in clusters.
• Demonstrated the method's applicability using labeled antibodies and Fab fragments against MHCI and MHCII.

Conclusions:

• The novel method enables simultaneous, cell-specific measurement of receptor rotational mobility and proximity.
• The technique provides a more comprehensive understanding of cell surface receptor organization and dynamics.
• This approach is adaptable to various instruments capable of dual-anisotropy detection, broadening its potential applications.