Two-Color Spatial Cumulant Analysis Detects Heteromeric Interactions between Membrane Proteins

Area of Science:

• Biophysics
• Cell Biology
• Spectroscopy

Background:

• Fluorescence fluctuation spectroscopy analyzes molecular aggregation using time-series data.
• Established methods like spatial intensity distribution analysis measure molecular brightness from single-color images.
• Analyzing molecular interactions often requires distinguishing between different fluorescently labeled molecules.

Purpose of the Study:

• To extend existing fluorescence fluctuation spectroscopy tools for analyzing two-color images.
• To resolve heteromeric interactions between spectrally distinct fluorescently labeled molecules.
• To develop and validate new methods for measuring molecular interactions in cellular environments.

Main Methods:

• Developed two-color spatial intensity distribution analysis and two-color spatial cumulant analysis (2c-SpCA).
• Implemented spectral shift filtering to remove cross-talk artifacts in hyperspectral imaging.
• Applied 2c-SpCA to images of living cells, including those with enhanced green fluorescent protein and mApple labeled heterodimers.

Main Results:

• 2c-SpCA demonstrated superior resolution for samples with multiple fluorescent species compared to other methods.
• Quantified the impact of resonance energy transfer and mApple maturation on brightness measurements.
• Successfully detected interactions between components of trimeric G-protein complexes using 2c-SpCA.

Conclusions:

• 2c-SpCA is a robust and computationally efficient method for measuring heteromeric interactions.
• The developed spectral shift filtering technique effectively removes artifacts in hyperspectral imaging.
• 2c-SpCA provides a valuable tool for studying molecular interactions in complex cellular systems.