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Analysis of Endocytic Trafficking by Single‐Cell Fluorescence Ratio Imaging

Herve Barriere1,  Gergely L. Lukacs1

1McGill 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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Abstract

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: Monitoring Synchronized Endocytic Trafficking of Transmembrane Proteins by Fluorescence Ratiometric Image Analysis of Vesicular pH
  • Alternate Protocol: 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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Materials

Basic Protocol: Monitoring Synchronized Endocytic Trafficking of Transmembrane Proteins by Fluorescence Ratiometric Image Analysis of Vesicular pH

 Materials
  • HEK 293 cells expressing CD4Tl and CD4Tl-Ub (Invitrogen; see Barrier et al., 2006 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% CO2
  • 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: Monitoring Post-Endocytic Trafficking of Transmembrane Proteins Following Continuous Labeling of Cargo with pH-Sensitive Fluorophore

 Additional Materials (also see Basic Protocol)
  • 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

 Additional Materials (also see Basic Protocol)
  • Prism4 software

Support Protocol 2: Measuring the pH of Recycling Endosomes

 Additional Materials (also see Basic Protocol)
  • 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

 Additional Materials (also see Basic Protocol)
  • 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)

 Additional Materials (also see Basic Protocol)
  • 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

 Additional Materials (also see Basic Protocol)
  • 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

 Additional Materials (also see Basic Protocol)
  • 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
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 15.13.1
    The lysosomal delivery kinetics of CD4Tl-Ub. Anti-CD4 and FITC-conjugated secondary Fab was bound to HEK 293 cells transfected with CD4Tl or CD4Tl-Ub for 1 hr at 0°C. The temperature was then raised to 37°C for 1, 15, or 30 min and the pHv was measured by FRIA. The mean pHv (±SEM) and the number of vesicles analyzed are indicated.

    View Image
  • Figure 15.13.2
    CD4TI-, CD4TI-Ub-, and CD4TI-UbR-expressing HEK 293 cells were allowed to internalize anti-CD4 primary Ab and FITC-conjugated secondary Fab for 1 hr at 37°C and chased for 30 min in Ab-free medium before FRIA. The mean pHv (±SEM) and the number of vesicles analyzed are indicated. Similar results were obtained in COS-7 cells.

    View Image
  • Figure 15.13.3
    Calibration curve using CD4Tl-Ub transfected HEK 293 cells. (A) Anti-CD4 primary Ab and FITC-conjugated secondary Fab are internalized for 1 hr at 37°C and chased for 30 min before FRIA. Calibration is performed as described in Support Protocol 1. (B) Distributions of fluorescence ratios of intracellular vesicles after clamping the cells at the indicated extracellular pH (pHe). (C) Measurement of antibody-cargo dissociation in acidic environment. The cell surface antibody binding is determined as described in Support Protocol 6 at the indicated extracellular pH. (D) Example of photobleaching control experiment. Fluorescence intensities at 495 nm and 440 nm excitation wavelengths and the measured ratio are recorded every 5 sec for 2-min period in HEK 293 CD4Tl-expressing cells. Internalization of Ab was performed as described in the Basic Protocol. Data were obtained on 25 vesicles (means ± SEM).

    View Image
  • Figure 15.13.4
    Colocalization and luminal pH determination studies for recycling endosomes, lysosomes, and the TGN. FRIA was carried out for recycling endosomes (A), lysosomes (B), and the TGN (C) using FITC-conjugated Tf, dextran, and FITC-Ab labeled CD4 as described in Support Protocols 2, 3 and 4 respectively. Subcellular colocalization of internalized CD4Tl (D), CD4Tl-Ub (E), and CD4T-TGN38 (F) chimeras with the respective organellar markers in HEK 293 cells was performed as described in the Support Protocol 5. Single optical sections were obtained by laser confocal fluorescence microscopy. Bar = 10 µm.

    View Image

Literature Cited

Literature Cited
    Barriere, H., Nemes, C., Lechardeur, D., Khan-Mohammad, M., Fruh, K., and Lukacs, G.L. 2006. Molecular basis of oligoubiquitin-dependent internalization of membrane proteins in Mammalian cells. Traffic. 7:282-297.
    Barriere, H., Nemes, C., Du, K., and Lukacs, G.L. 2007. Plasticity of Poly-Ubiquitin Recognition as Lysosomal Targeting Signals by the Endosomal Sorting Machinery. Mol. Biol. Cell. 18:3952-3965.
    Bonifacino, J.S. and Rojas, R. 2006. Retrograde transport from endosomes to the trans-Golgi network. Nat. Rev. Mol. Cell Biol.. 7:568-579.
    Bonifacino, J.S. and Traub, L.M 2003. Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72:395-447.
    Bosshart, H., Humphrey, J., Deignan, E., Davidson, J., Drazba, J., Yuan, L., Oorschot, V., Peters, P.J., and Bonifacino, J.S. 1994. The cytoplasmic domain mediates localization of furin to the trans-Golgi network en route to the endosomal/lysosomal system. J. Cell Biol. 126:1157-1172.
    Davis, C.G., Goldstein, J.L., Sudhof, T.C., Anderson, R.G., Russell, D.W., and Brown, M.S. 1987. Acid-dependent ligand dissociation and recycling of LDL receptor mediated by growth factor homology region. Nature. 326:760-765.
    Delmotte, C. and Delmas, A. 1999. Synthesis and fluorescence properties of Oregon Green 514 labeled peptides. Bioorg. Med. Chem. Lett. 9:2989-2994.
    Demaurex, N., Furuya, W., D'Souza, S., Bonifacino, J.S., and Grinstein, S. 1998. Mechanism of acidification of the trans-Golgi network (TGN). In situ measurements of pH using retrieval of TGN38 and furin from the cell surface. J. Biol. Chem. 273:2044-2051.
    DiPaola, M. and F.R. Maxfield. 1984. Conformational changes in the receptors for epidermal growth factor and asialoglycoproteins induced by the mildly acidic pH found in endocytic vesicles. J. Biol. Chem. 259:9163-9171.
    Erdahl, W.L., Chapman, C.J., Taylor, R.W., and Pfeiffer, D.R. 1995. Effects of pH conditions on Ca2 +transport catalyzed by ionophores A23187, 4-BrA23187, and ionomycin suggest problems with common applications of these compounds in biological systems. Biophys. J.. 69:2350-2363.
    Falcon-Perez, J.M., Nazarian, R., Sabatti, C., and Dell'Angelica, E.C. 2005. Distribution and dynamics of Lamp1-containing endocytic organelles in fibroblasts deficient in BLOC-3. J. Cell Sci. 118:5243-5255.
    Farinas, J. and Verkman, A.S. 1999. Receptor-mediated targeting of fluorescent probes in living cells. J. Biol. Chem. 274:7603-7606.
    Geisow, M.J. 1984. Fluorescein conjugates as indicators of subcellular pH. A critical evaluation. Exp. Cell Res. 150:29-35.
    Ghosh, P., Dahms, N.M., and Kornfeld, S. 2003. Mannose 6-phosphate receptors: New twists in the tale. Nat. Rev. Mol. Cell Biol. 4:202-212.
    Harold, F.M. and Baarda, J.R. 1968. Effects of nigericin and monactin on cation permeability of Streptococcus faecalis and metabolic capacities of potassium-depleted cells. J. Bacteriol. 95:816-823.
    Humphrey, J.S., Peters, P.J., Yuan, L.C., and Bonifacino, J.S. 1993. Localization of TGN38 to the trans-Golgi network: Involvement of a cytoplasmic tyrosine-containing sequence. J. Cell Biol. 120:1123-1135.
    Janvier, K. and Bonifacino, J.S. 2005. Role of the endocytic machinery in the sorting of lysosome-associated membrane proteins. Mol. Biol. Cell. 16:4231-4242.
    Katzmann, D.J., Odorizzi, G., and Emr, S.D.. 2002. Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell Biol. 3:893-905.
    Kim, J.H., Lingwood, C.A., Williams, D.B., Furuya, W., Manolson, M.F., and Grinstein, S. 1996. Dynamic measurement of the pH of the Golgi complex in living cells using retrograde transport of the verotoxin receptor. J. Cell Biol. 134:1387-1399.
    Lencer, W.I., Weyer, P., Verkman, A.S., Ausiello, D.A., and Brown, D. 1990. FITC-dextran as a probe for endosome function and localization in kidney. Am. J. Physiol. 258:C309-C317.
    Llopis, J., McCaffery, J.M., Miyawaki, A., Farquhar, M.G., and Tsien, R.Y. 1998. Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc. Natl. Acad. Sci. U.S.A. 95:6803-6808.
    Loffing, J., Moyer, B.D., Reynolds, D., and Stanton, B.A.. 1999. PBA increases CFTR expression but at high doses inhibits Cl(-) secretion in Calu-3 airway epithelial cells. Am. J. Physiol. 277:L700-L708.
    Lukacs, G.L., Segal, G., Kartner, N., Grinstein, S., and Zhang, F. 1997. Constitutive internalization of cystic fibrosis transmembrane conductance regulator occurs via clathrin-dependent endocytosis and is regulated by protein phosphorylation. Biochem. J.. 328:353-361.
    Machen, T.E., Leigh, M.J., Taylor, C., Kimura, T., Asano, S., and Moore, H.P. 2003. pH of TGN and recycling endosomes of H+/K+-ATPase-transfected HEK-293 cells: Implications for pH regulation in the secretory pathway. Am. J. Physiol. Cell Physiol.. 285:C205-C214.
    Mallet, W.G. and Maxfield, F.R. 1999. Chimeric forms of furin and TGN38 are transported with the plasma membrane in the trans-Golgi network via distinct endosomal pathways. J. Cell Biol. 146:345-359.
    Marks, M.S., Woodruff, L., Ohno, H., and Bonifacino, J.S.. 1996. Protein targeting by tyrosine- and di-leucine-based signals: Evidence for distinct saturable components. J. Cell Biol. 135:341-354.
    Maxfield, F.R. 1982. Weak bases and ionophores rapidly and reversibly raise the pH of endocytic vesicles in cultured mouse fibroblasts. J. Cell Biol. 95:676-681.
    Maxfield, F.R. 1989. Measurement of vacuolar pH and cytoplasmic calcium in living cells using fluorescence microscopy. Methods Enzymol. 173:745-771.
    Maxfield, F.R. and McGraw, T.E. 2004. Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5:121-132.
    Molloy, S.S., Thomas, L., VanSlyke, J.R., Stenberg, P.E., and Thomas, G. 1994. Intracellular trafficking and activation of the furin proprotein convertase: localization to the TGN and recycling from the cell surface. EMBO J. 13:18-33.
    Moyer, B.D., Loffing-Cueni, D., Loffing, J., Reynolds, D., and Stanton, B.A. 1999. Butyrate increases apical membrane CFTR but reduces chloride secretion in MDCK cells. Am. J. Physiol. 277:F271-F276.
    Mukherjee, S., Ghosh, R.N., and Maxfield, F.R. 1997. Endocytosis. Physiol. Rev. 77:759-803.
    Ohkuma, S. and Poole, B. 1978. Fluorescence probe measurement of the intralysosomal pH in living cells and the perturbation of pH by various agents. Proc. Natl. Acad. Sci. U.S.A. 75:3327-3331.
    Piguet, V., Gu, F., Foti, M., Demaurex, N., Gruenberg, J., Carpentier, J.L., and Trono, D. 1999. Nef-induced CD4 degradation: A diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes. Cell. 97:63-73.
    Poet, M., Kornak, U., Schweizer, M., Zdebik, A.A., Scheel, O., Hoelter, S., Wurst, W., Schmitt, A., Fuhrmann, J.C., Planells-Cases, R., Mole, S.E., Hubner, C.A., and. Jentsch, T.J. 2006. Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6. Proc. Natl. Acad. Sci. U.S.A. 103:13854-13859.
    Presley, J.F., Mayor, S., Dunn, K.W., Johnson, L.S., McGraw, T.E., and Maxfield, F.R. 1993. The End2 mutation in CHO cells slows the exit of transferrin receptors from the recycling compartment but bulk membrane recycling is unaffected. J. Cell Biol. 122:1231-1241.
    Seksek, O., Biwersi, J., and Verkman, A.S. 1995. Direct measurement of trans-Golgi pH in living cells and regulation by second messengers. J. Biol. Chem. 270:4967-4970.
    Sharma, D.K., Choudhury, A., Singh, R.D., Wheatley, C.L., Marks, D.L., and Pagano, R.E. 2003. Glycosphingolipids internalized via caveolar-related endocytosis rapidly merge with the clathrin pathway in early endosomes and form microdomains for recycling. J. Biol. Chem. 278:7564-7572.
    Sharma, M., Pampinella, F., Nemes, C., Benharouga, M., So, J., Du, K., Bache, K.G., Papsin, B., Zerangue, N., Stenmark, H., and Lukacs, G.L. 2004. Misfolding diverts CFTR from recycling to degradation: Quality control at early endosomes. J. Cell Biol. 164:923-933.
    Subtil, A., Hemar, A., and Dautry-Varsat, A. 1994. Rapid endocytosis of interleukin 2 receptors when clathrin-coated pit endocytosis is inhibited. J. Cell Sci. 107:3461-3468.
    Sun-Wada, G.H., Wada, Y., and Futai, M. 2004. Diverse and essential roles of mammalian vacuolar-type proton pump ATPase: Toward the physiological understanding of inside acidic compartments. Biochim. Biophys. Acta. 1658:106-114.
    Tartakoff, A., Vassalli, P., and Detraz, M. 1978. Comparative studies of intracellular transport of secretory proteins. J. Cell Biol. 79:694-707.
    Tycko, B. and Maxfield, F.R. 1982. Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin. Cell. 28:643-51.
    von Zastrow, M. and Sorkin, A. 2007. Signaling on the endocytic pathway. Curr. Opin. Cell Biol. 19:436-445.
    Weissman, A.M., Klausner, R.D., Rao, K., and Harford, J.B.. 1986. Exposure of K562 cells to anti-receptor monoclonal antibody OKT9 results in rapid redistribution and enhanced degradation of the transferrin receptor. J. Cell Biol. 102:951-958.
    Wu, M.M., Llopis, J., Adams, S., McCaffery, J.M., Kulomaa, M.S., Machen, T.E., Moore, H.P., and Tsien, R.Y. 2000. Organelle pH studies using targeted avidin and fluorescein-biotin. Chem. Biol. 7:197-209.
    Wu, M.M., Grabe, M., Adams, S., Tsien, R.Y., Moore, H.P., and Machen, T.E. 2001. Mechanisms of pH regulation in the regulated secretory pathway. J. Biol. Chem. 276:33027-33035.
    Yamashiro, D.J., Tycko, B., Fluss, S.R., and Maxfield, F.R. 1984. Segregation of transferrin to a mildly acidic (pH 6.5) para-Golgi compartment in the recycling pathway. Cell. 37:789-800.
     
 
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