Analysis of Endocytic Trafficking by Single‐Cell Fluorescence Ratio Imaging

Herve Barriere1, Gergely L. Lukacs1

1 McGill University, Department of Physiology, Montreal, Quebec, Canada
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 15.13
DOI:  10.1002/0471143030.cb1513s40
Online Posting Date:  September, 2008
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The post‐endocytic sorting of internalized membrane proteins plays a critical role in numerous physiological processes, including receptor desensitization, degradation of non‐native plasma membrane proteins, and cell surface retrieval of receptors from early endosomes upon ligand dissociation. Here, we describe a fluorescence ratiometric image analysis (FRIA) method used to determine the post‐endocytic fate and transport kinetics of transmembrane proteins based on the pH measurement of internalized cargo‐containing compartments in living cells. The method relies on the notion that the pH of a cargo‐containing transport vesicle (vesicular pH, pHv) could be taken as an indicator of its identity, considering that endocytic organelles (e.g., sorting endosome, recycling endosome, late endosome/MVB, and lysosome) have characteristic pHv. The pH‐sensitive FITC‐conjugated secondary antibody is attached to the cargo via a primary antibody, recognizing the cargo extracellular domain. The pHv is determined by single‐cell FRIA. Internalized cargo colocalization with organellar markers, as well as pHv measurement of recycling endosome, lysosome, and the TGN are discussed to validate the technique and facilitate data interpretation. Curr. Protoc. Cell Biol. 40:15.13.1‐15.13.21. © 2008 by John Wiley & Sons, Inc.

Keywords: post‐endocytotic sorting; pH; fluorescence ratiometric image analysis; endocytic organelles

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Table of Contents

  • Introduction
  • Basic Protocol 1: Monitoring Synchronized Endocytic Trafficking of Transmembrane Proteins by Fluorescence Ratiometric Image Analysis of Vesicular pH
  • Alternate Protocol 1: Monitoring Post‐Endocytic Trafficking of Transmembrane Proteins Following Continuous Labeling of Cargo with pH‐Sensitive Fluorophore
  • Support Protocol 1: Performing Multi‐Point in Situ pH Calibration
  • Support Protocol 2: Measuring the pH of Recycling Endosomes
  • Support Protocol 3: Measuring the pH of Lysosomes
  • Support Protocol 4: Measuring the pH of the Trans‐Golgi Network (TGN)
  • Support Protocol 5: Verifying the Post‐Endocytic Fate of Internalized Membrane Protein by Immunolocalization
  • Support Protocol 6: Determining the Antibody‐Cargo Complex Stability in Acidic Environments
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Monitoring Synchronized Endocytic Trafficking of Transmembrane Proteins by Fluorescence Ratiometric Image Analysis of Vesicular pH

  • HEK 293 cells expressing CD4Tl and CD4Tl‐Ub (Invitrogen; see Barrier et al., for a description of cell lines)
  • DMEM‐F: DMEM supplemented with 10% (v/v) fetal bovine serum (FBS)
  • COS‐7 cells (ATCC, CRL‐1651), optional
  • Fugene 6 (Roche)
  • H‐DMEM‐F: DMEM without bicarbonate and phenol‐red (Invitrogen) supplemented with 5% (v/v) bovine serum (BS) and 15 mM HEPES
  • Anti‐CD4 antibody OKT4 (Harlan Bioproducts)
  • FITC‐conjugated goat anti–mouse secondary Fab (Jackson ImmunoResearch Laboratories)
  • PBS++ solution (see recipe)
  • Modified Ringer's solution (see recipe)
  • K+‐rich buffer (see recipe)
  • Nigericin (Sigma‐Aldrich, cat. no. N‐7143)
  • Monensin (Sigma‐Aldrich, cat. no. M‐5273)
  • 25‐mm glass coverslips with standard no. 1 or no. 1.5 thickness, sterile, coated with poly‐L‐lysine
  • 6‐well culture plates (Falcon, cat. no. 353046)
  • Tissue culture incubator at 37°C with 5% CO 2
  • 35‐mm tissue culture dish
  • Perfusion chamber (MSC‐TD, Warner Instruments)
  • Thermostated coverslip holder: Bipolar temperature controller (Med Systems, TC‐202)
  • Microscope: inverted fluorescence microscope (e.g., Zeiss Axiovert 100 TV) equipped with:
    • Planachromat objective (63× NA 1.4, Carl Zeiss MicroImaging)
    • Objective heater (TempControl mini, Carl Zeiss MicroImaging)
    • Cooled CCD camera (e.g., Hamamatsu ORCA‐ER CCD)
    • Filter set for BCECF: excitation filters, D495/10 and D440/20 and emission filter, D535/25 (Chroma)
    • Lambda 10‐2 filter wheel (Sutter Instrument), containing the D495/10 and D440/20 excitation filters and used during image acquisition in step 10
  • Lamp: X‐Cite 120 fluorescence illumination system (EXFO) allowing the adjustment of the Hg‐lamp intensity (EXFO Life Sciences Division)
  • MetaFluor (MDS Analytical Technologies) Imaging System (for ratiometric fluorescence ion measurements and run on a PC computer)
  • OriginPro7 software
  • Ice bucket [used for cooling the cells at 4°C (e.g., steps 2, 3, 5, and 8)]
  • Vacuum and perfusion [used for replacing medium (see step 11)]

Alternate Protocol 1: Monitoring Post‐Endocytic Trafficking of Transmembrane Proteins Following Continuous Labeling of Cargo with pH‐Sensitive Fluorophore

  • COS‐7 or HEK 293 cells, 70% confluent in 60‐mm dishes
  • pCDNA3 expression vector encoding CD4Tl, CD4Tl‐Ub, or CD4Tl‐UbR
  • Fugene 6 (Roche)

Support Protocol 1: Performing Multi‐Point in Situ pH Calibration

  • Prism4 software

Support Protocol 2: Measuring the pH of Recycling Endosomes

  • FITC‐Transferrin f (FITC‐Tf; Molecular Probes)
  • Holotransferrin Sigma, cat. no T‐0665)
  • Ovalbumin (Sigma, cat. no. A‐5503)

Support Protocol 3: Measuring the pH of Lysosomes

  • FITC‐Dextran (mol. wt. 10 kDa anionic; Molecular Probes)
  • Dextran (mol. wt. 68.8 kDa; cat. no. D4876)

Support Protocol 4: Measuring the pH of the Trans‐Golgi Network (TGN)

  • Expression plasmid encoding the CD4T‐TGN38 chimera, pCDNA3.1 (Invitrogen)
  • Fugene 6 (Roche)

Support Protocol 5: Verifying the Post‐Endocytic Fate of Internalized Membrane Protein by Immunolocalization

  • Antibodies:
    • Anti‐TGN46 (AHP500; AbD Serotec)
    • TRITC‐conjugated goat anti‐mouse Fab (Jackson ImmunoResearch Laboratories)
  • TRITC‐Tf (Jackson ImmunoResearch Laboratories)
  • 4% (v/v) paraformaldehyde solution (see recipe)
  • 1 M glycine containing PBS++ solution (see recipe)
  • Mounting medium (e.g., Vectashield H‐1200, Vector Laboratories)
  • Laser confocal fluorescence microscope (e.g., LSM 510 Carl Zeiss)

Support Protocol 6: Determining the Antibody‐Cargo Complex Stability in Acidic Environments

  • HRP‐conjugated anti–mouse Fab antibody (Jackson ImmunoResearch Laboratories)
  • Acetic acid buffer adjusted to pH 2.5 and 5.0
  • Recovery buffer (see recipe)
  • AmplexRed kit (Molecular Probes, cat. no. A22170)
  • BCA assay kit (BioRad)
  • 12‐well culture plates
  • 96‐well black plates (Greiner)
  • Fluorescent plate reader (e.g., POLARstar OPTIMA; BMG Labtech) with 560 and 590 nm excitation and emission wavelengths, respectively
  • Spectrophotometer
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