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Quantifying receptor trafficking and colocalization with confocal microscopy.

Jeremy A Pike1, Iain B Styles2, Joshua Z Rappoport3

  • 1PSIBS Doctoral Training Centre, School of Chemistry, University of Birmingham, Birmingham B15 2TT, UK; School of Biosciences, University of Birmingham, Birmingham B15 2TT, UK; Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge CB2 0RE, UK.

Methods (San Diego, Calif.)
|January 30, 2017
PubMed
Summary
This summary is machine-generated.

This study presents new image analysis methods for tracking cellular receptor trafficking using confocal microscopy. Aberrant epidermal growth factor receptor (EGFR) trafficking was significantly reduced in treated cells.

Keywords:
ColocalizationConfocalEGFREndocytosisReceptorTrafficking

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Confocal microscopy is vital for studying cellular processes like receptor trafficking and endocytosis.
  • Robust image analysis is crucial for identifying and studying abnormal trafficking patterns.
  • Existing methods may lack the precision needed for complex live-cell 3D time-lapse data.

Purpose of the Study:

  • To develop and present custom, unbiased image analysis workflows for live-cell 3D time-lapse data.
  • To quantify changes in subcellular receptor distribution and endocytosis.
  • To provide a quantitative method for assessing receptor-endosome colocalization.

Main Methods:

  • Utilized 3D level set segmentation for accurate cell boundary identification based on fluorescently labeled receptors.
  • Developed protocols for pre-processing live-cell 3D time-lapse data, including denoising and background subtraction.
  • Employed Manders and Pearson coefficients to quantitatively analyze receptor-endosome colocalization.

Main Results:

  • Demonstrated a significant reduction in ligand-stimulated epidermal growth factor receptor (EGFR) trafficking in AG1478 and Dynasore treated cells.
  • Quantified changes in subcellular receptor distribution over time.
  • Showed a statistically significant decrease in EGFR and rab5-positive endosome co-occurrence in treated cells compared to controls.

Conclusions:

  • The developed image analysis workflows provide robust and unbiased quantification of cellular receptor trafficking and endocytosis.
  • The methods enable accurate assessment of aberrant trafficking, as exemplified by EGFR.
  • The study offers a new visualization strategy for receptor-endosome co-occurrence, improving upon traditional color overlays.