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Fluorescence Intensity of Protein Tags Is Dependent on Their Subcellular Location.

Yan Chen1, John P Eichorst1, Barbara Barylko2

  • 1School of Physics and Astronomy, University of Minnesota, Minneapolis, MN 55455, USA.

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|February 26, 2026
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Summary

Fluorescent protein (FP) tagging can alter apparent protein localization due to pH-dependent fluorescence. Different FP tags, like EGFP and mCherry, show surprising distribution discrepancies on organelle surfaces, indicating microenvironmental sensitivity.

Keywords:
EGFPdifferential fluorescence intensitiesfluorescent proteins (FPs)mCherrysecretory/endocytic pathway

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Area of Science:

  • Cell Biology
  • Biochemistry
  • Molecular Imaging

Background:

  • Fluorescent protein (FP) tagging is crucial for visualizing protein localization in cells.
  • FP fluorescence intensity is pH-dependent due to chromophore protonation, affecting quantitative analysis.
  • The pKa of an FP influences its brightness at different cellular pH levels.

Purpose of the Study:

  • To investigate how different FP tags affect the perceived subcellular distribution of proteins on organelle surfaces.
  • To determine if FP pKa influences apparent protein localization in the cytoplasm.
  • To assess the impact of FP microenvironmental sensitivity on imaging studies.

Main Methods:

  • Utilized EGFP and mCherry fluorescent proteins for tagging cellular proteins.
  • Examined proteins localized to the cytoplasmic surfaces of secretory/endocytic organelles.
  • Quantified signal overlap using Pearson's correlation coefficients for different FP tag combinations.

Main Results:

  • Observed significant discrepancies in apparent protein distribution when comparing EGFP- and mCherry-tagged proteins (PCC ~0.80).
  • Discrepancies were minimal when comparing mCherry with low pKa FPs like mTurquoise or mCerulean (PCC ~0.90-0.95).
  • These differences suggest FP tags are sensitive to organelle surface microenvironments, not just bulk cytoplasmic pH.

Conclusions:

  • FP tags can exhibit differential behavior on organelle surfaces, impacting perceived protein localization.
  • The choice of FP tag is critical and can introduce artifacts in subcellular distribution studies.
  • Microenvironmental factors around organelles may influence FP characteristics beyond simple pH effects.