Quantifying Protein Copy Number in Super Resolution Using an Imaging-Invariant Calibration

Area of Science:

• Cellular Biology
• Biophysics
• Microscopy

Background:

• Super-resolution microscopy reveals proteins form cellular nanoclusters.
• Quantifying protein copy number in nanoclusters is difficult due to labeling and fluorophore complexities.
• Previous DNA-origami calibration methods were condition-specific, requiring repeated experiments.

Purpose of the Study:

• To develop a versatile calibration method for protein copy number quantification in super-resolution microscopy.
• To extend previously established DNA-origami calibration data to new experimental conditions.
• To enable quantification of any green-fluorescent-protein-fused protein.

Main Methods:

• Utilized HeLa cells stably expressing dynein intermediate chain fused to green fluorescent protein (HeLa IC74 cells) as a reference sample.
• Quantified dynein motor copy number within nanoclusters in the cytosol and along microtubules.
• Extended existing DNA-origami-based calibration data to new imaging and labeling conditions.

Main Results:

• Demonstrated that previously generated DNA-origami calibration data can be applied to different experimental conditions.
• Successfully quantified dynein motor copy number in nanoclusters, revealing assemblies of multiple motors, particularly along microtubules.
• Established HeLa IC74 cells as a reference sample for condition-independent protein copy number calibration.

Conclusions:

• The developed approach significantly enhances the versatility and applicability of protein copy number quantification in super-resolution microscopy.
• Protein nanocluster analysis is now feasible across varied experimental setups without re-calibration.
• This method facilitates a deeper understanding of protein organization and function within cellular nanoclusters.