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Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells
Published on: June 30, 2018
50nm-scale localization of single unmodified, isotopically enriched, proteins in cells
Anthony Delaune1, Armelle Cabin-Flaman, Guillaume Legent
1Laboratoire MERCI EA3829, équipe AMMIS, Faculté des Sciences de l'Université de Rouen, Mont Saint Aignan, France. anthony.delaune@univ-rouen.fr
Plos One
|February 23, 2013
Summary
This study introduces a new algorithm for imaging single proteins within cells using dynamic Secondary Ion Mass Spectrometry (D-SIMS). The method accurately detects and localizes (15)N-enriched proteins, even down to a single molecule, avoiding artifacts.
Area of Science:
- Cellular Biology
- Biophysics
- Analytical Chemistry
Background:
- Imaging single proteins in cells is difficult without introducing artifacts from tagging or antibodies.
- Isotopic enrichment with Nitrogen-15 ((15)N) is believed to preserve protein function.
- Dynamic Secondary Ion Mass Spectrometry (D-SIMS) can localize (15)N-enriched proteins.
Purpose of the Study:
- To develop a novel imaging analysis algorithm for detecting and quantifying single (15)N-enriched proteins within cells using D-SIMS.
- To overcome the limitations of existing protein imaging techniques by avoiding tagging or antibody-based methods.
- To achieve high-resolution (50 nm) localization of individual unmodified proteins.
Main Methods:
- Utilized dynamic Secondary Ion Mass Spectrometry (D-SIMS) for isotopic analysis.
- Developed a statistical algorithm to differentiate (15)N-enriched protein signals from background noise.
- Employed sequential D-SIMS images to compare measured isotopic fractions with expected values for 1, 2, or more enriched proteins to determine protein count.
- Applied the method to localize (15)N-enriched thymine DNA glycosylase (TDG) and retinoid X receptor α (RXRα) in COS-7 cells.
Main Results:
- Successfully detected and localized single (15)N-enriched thymine DNA glycosylase (TDG) and retinoid X receptor α (RXRα) proteins.
- Observed proteins primarily in the nucleus after 4 hours, with RXRα as monomer/dimer and TDG as monomer.
- After 7 hours, RXRα was found in the nucleus (monomer, dimer, tetramer), while TDG formed cytoplasmic clusters.
- After 24 hours, RXRα formed cytoplasmic clusters, and TDG was no longer detectable.
Conclusions:
- Single unmodified proteins can be accurately counted and localized within cells using D-SIMS combined with the developed analysis algorithm.
- The method provides a resolution of 50 nm, enabling detailed study of protein dynamics.
- This technique offers a powerful, artifact-free approach for investigating the behavior of individual proteins in cellular environments.
