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Endocytic Sorting of CFTR Variants Monitored by Single-Cell Fluorescence Ratiometric Image Analysis (FRIA) in Living Cells

  • Herve Barrière
  • Pirjo Apaja
  • Tsukasa Okiyoneda
  • Gergely L. LukacsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 741)

Abstract

The wild-type CFTR channel undergoes constitutive internalization and recycling at the plasma membrane. This process is initiated by the recognition of the Tyr- and di-Leu-based endocytic motifs of CFTR by the AP-2 adaptor complex, leading to the formation of clathrin-coated vesicles and the channel delivery to sorting/recycling endosomes. Accumulating evidence suggests that conformationally defective mutant CFTRs (e.g. rescued F508del and glycosylation-deficient channel) are unstable at the plasma membrane and undergo augmented ubiquitination in post-Golgi compartments. Ubiquitination conceivably accounts for the metabolic instability at cell surface by provoking accelerated internalization, as well as rerouting the channel from recycling towards lysosomal degradation. We developed an in vivo fluorescence ratiometric image analysis (FRIA) that in concert with genetic manipulation can be utilized to establish the post-endocytic fate and sorting determinants of mutant CFTRs.

Key words

CF mutations conformational defect endosomal sorting recycling lysosomal targeting ubiquitin-binding protein plasma membrane ESCRT vesicular pH siRNA 

References

  1. 1.
    Mukherjee, S., Ghosh, R., and Maxfield, F. (1997) Endocytosis. Physiol. Rev. 77, 759–803.PubMedGoogle Scholar
  2. 2.
    Sharma, M., Pampinella, F., Nemes, C., Benharouga, M., So, J., Du, K., et al. (2004) Misfolding diverts CFTR from recycling to degradation: quality control at early endosomes. J. Cell Biol. 164, 923–933.PubMedCrossRefGoogle Scholar
  3. 3.
    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.PubMedCrossRefGoogle Scholar
  4. 4.
    Bonifacino, J. S., and Traub, L. M. (2003) Signals for sorting of transmembrane proteins to endosomes and lysosomes. Annu. Rev. Biochem. 72, 395–447.PubMedCrossRefGoogle Scholar
  5. 5.
    Katzmann, D. J., Odorizzi, G., and Emr, S. D. (2002) Receptor downregulation and multivesicular-body sorting. Nat. Rev. Mol. Cell Biol. 3, 893–905.PubMedCrossRefGoogle Scholar
  6. 6.
    Maxfield, F. R., and McGraw, T. E. (2004) Endocytic recycling. Nat. Rev. Mol. Cell Biol. 5, 121–132.PubMedCrossRefGoogle Scholar
  7. 7.
    von Zastrow, M., and Sorkin, A. (2007) Signaling on the endocytic pathway. Curr. Opin. Cell Biol. 19, 436–445.CrossRefGoogle Scholar
  8. 8.
    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.PubMedCrossRefGoogle Scholar
  9. 9.
    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.PubMedCrossRefGoogle Scholar
  10. 10.
    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.PubMedCrossRefGoogle Scholar
  11. 11.
    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.PubMedCrossRefGoogle Scholar
  12. 12.
    Bonifacino, J. S., and Rojas, R. (2006) Retrograde transport from endosomes to the trans-Golgi network. Nat. Rev. Mol. Cell Biol. 7, 568–579.PubMedCrossRefGoogle Scholar
  13. 13.
    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.PubMedGoogle Scholar
  14. 14.
    Weixel, K. M., and Bradbury, N. A. (2001) Mu 2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin-mediated endocytic pathway. J. Biol. Chem. 276, 46251–46259.PubMedCrossRefGoogle Scholar
  15. 15.
    Gentzsch, M., Chang, X. B., Cui, L., Wu, Y., Ozols, V. V., Choudhury, A., et al. (2004) Endocytic trafficking routes of wild type and DeltaF508 cystic fibrosis transmembrane conductance regulator. Mol. Biol. Cell 15, 2684–2696.PubMedCrossRefGoogle Scholar
  16. 16.
    Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., et al. (1999) Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator requires a tyrosine-based signal. J. Biol. Chem. 274, 3602–3609.PubMedCrossRefGoogle Scholar
  17. 17.
    Swiatecka-Urban, A., Duhaime, M., Coutermarsh, B., Karlson, K. H., Collawn, J., Milewski, M., et al. (2002) PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 277, 40099–40105.PubMedCrossRefGoogle Scholar
  18. 18.
    Swiatecka-Urban, A., Boyd, C., Coutermarsh, B., Karlson, K. H., Barnaby, R., Aschenbrenner, L., et al. (2004) Myosin VI regulates endocytosis of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 279, 38025–38031.PubMedCrossRefGoogle Scholar
  19. 19.
    Picciano, J. A., Ameen, N., Grant, B. D., and Bradbury, N. A. (2003) Rme-1 regulates the recycling of the cystic fibrosis transmembrane conductance regulator. Am. J. Physiol. Cell Physiol. 285, C1009–C1018.PubMedGoogle Scholar
  20. 20.
    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. USA 75, 3327–3331.PubMedCrossRefGoogle Scholar
  21. 21.
    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.PubMedCrossRefGoogle Scholar
  22. 22.
    Glozman, R., Okiyoneda, T., Mulvihill, C. M., Rini, J. M., Barriere, H., and Lukacs, G. L. (2009) N-glycans are direct determinants of CFTR folding and stability in secretory and endocytic membrane traffic. J. Cell Biol. 184, 847–862.PubMedCrossRefGoogle Scholar
  23. 23.
    Barriere, H., Bagdany, M., Bossard, F., Okiyoneda, T., Wojewodka, G., Gruenert, D., et al. (2009) Revisiting the role of cystic fibrosis transmembrane conductance regulator and counterion permeability in the pH regulation of endocytic organelles. Mol. Biol. Cell 20, 3125–3141.PubMedCrossRefGoogle Scholar
  24. 24.
    Geisow, M. J. (1984) Fluorescein conjugates as indicators of subcellular pH. A critical evaluation. Exp. Cell Res. 150, 29–35.PubMedCrossRefGoogle Scholar
  25. 25.
    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.PubMedGoogle Scholar
  26. 26.
    Tartakoff, A., Vassalli, P., and Detraz, M. (1978) Comparative studies of intracellular transport of secretory proteins. J. Cell Biol. 79, 694–707.PubMedCrossRefGoogle Scholar
  27. 27.
    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.PubMedCrossRefGoogle Scholar
  28. 28.
    Kumar, K. G., Barriere, H., Carbone, C. J., Liu, J., Swaminathan, G., Xu, P., et al. (2007) Site-specific ubiquitination exposes a linear motif to promote interferon-alpha receptor endocytosis. J. Cell Biol. 179, 935–950.PubMedCrossRefGoogle Scholar
  29. 29.
    Barriere, H., and Lukacs, G. L. (2008) Analysis of endocytic trafficking by single-cell fluorescence ratio imaging. Curr. Protoc. Cell. Biol. Ch. 15, Unit 15 13.1–13.21.Google Scholar
  30. 30.
    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.PubMedGoogle Scholar
  31. 31.
    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.PubMedCrossRefGoogle Scholar
  32. 32.
    Delmotte, C., and Delmas, A. (1999) Synthesis and fluorescence properties of Oregon Green 514 labeled peptides. Bioorg. Med. Chem. Lett. 9, 2989–2994.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Herve Barrière
    • 1
  • Pirjo Apaja
    • 1
  • Tsukasa Okiyoneda
    • 1
  • Gergely L. Lukacs
    • 1
    Email author
  1. 1.Department of PhysiologyMcGill UniversityMontréalCanada

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