Advertisement

Quantitative Analysis of Transferrin Cycling by Automated Fluorescence Microscopy

  • David T. Hirschmann
  • Christoph A. Kasper
  • Martin Spiess
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1270)

Abstract

Surface receptors are transported between the plasma membrane and intracellular compartments by various endocytic mechanisms and by recycling via different pathways from sorting or recycling endosomes. The analysis of cellular components involved in mediating or regulating these transport steps is of high current interest and requires quantitative methods to determine rates of endocytosis and/or recycling. Various biochemical procedures to measure uptake of labeled ligand molecules or internalization and reappearance of surface-labeled receptors have been developed. Here, we describe a quantitative method based on fluorescence microscopy of adherent cells taking advantage of the transferrin (Tf) receptor as the prototype of cycling transport receptors. Tf is endocytosed with bound Fe3+ and, upon release of the iron ion in endosomes, recycled as apo-Tf together with the receptor. To follow the ligand–receptor complex, fluorescently labeled Tf is used and detected microscopically with or without releasing Tf from cell surface receptors by acid stripping. To go beyond the observation of a few individual cells, automated fluorescence microscopy is employed to image thousands of cells at different time points and in parallel with different treatments (such as chemical inhibitors, siRNA silencing, or transfection of candidate genes) in a 96-well format. Computer-assisted image analysis allows unbiased quantitation of Tf content of each cell and to distinguish between different cell populations.

Key words

Endocytosis Fluorescence microscopy Image analysis Receptor Recycling Transferrin 

Notes

Acknowledgments

Our work was supported by grant 31003A-125423 from the Swiss National Science Foundation. We are grateful to Dr. Cécile Arrieumerlou (Biozentrum, University of Basel) for her support.

References

  1. 1.
    Schmid EM, McMahon HT (2007) Integrating molecular and network biology to decode endocytosis. Nature 448:883–888CrossRefPubMedGoogle Scholar
  2. 2.
    Howes MT, Mayor S, Parton RG (2010) Molecules, mechanisms, and cellular roles of clathrin-independent endocytosis. Curr Opin Cell Biol 22:519–527CrossRefPubMedGoogle Scholar
  3. 3.
    Sandvig K, Pust S, Skotland T, van Deurs B (2011) Clathrin-independent endocytosis: mechanisms and function. Curr Opin Cell Biol 23:413–420CrossRefPubMedGoogle Scholar
  4. 4.
    Maldonado-Baez L, Williamson C, Donaldson JG (2013) Clathrin-independent endocytosis: a cargo-centric view. Exp Cell Res 319:2759–2769CrossRefPubMedCentralPubMedGoogle Scholar
  5. 5.
    Maxfield FR, McGraw TE (2004) Endocytic recycling. Nat Rev Mol Cell Biol 5:121–132CrossRefPubMedGoogle Scholar
  6. 6.
    Grant BD, Donaldson JG (2009) Pathways and mechanisms of endocytic recycling. Nat Rev Mol Cell Biol 10:597–608CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Ciechanover A, Schwartz AL, Dautry-Varsat A, Lodish HF (1983) Kinetics of internalization and recycling of transferrin and the transferrin receptor in a human hepatoma cell line. Effect of lysosomotropic agents. J Biol Chem 258:9681–9689PubMedGoogle Scholar
  8. 8.
    Stein BS, Sussman HH (1986) Demonstration of two distinct transferrin receptor recycling pathways and transferrin-independent receptor internalization in K562 cells. J Biol Chem 261:10319–10331PubMedGoogle Scholar
  9. 9.
    Klausner RD, Van Renswoude J, Ashwell G, Kempf C, Schechter AN, Dean A, Bridges KR (1983) Receptor-mediated endocytosis of transferrin in K562 cells. J Biol Chem 258:4715–4724PubMedGoogle Scholar
  10. 10.
    Davis RJ, Corvera S, Czech MP (1986) Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane. J Biol Chem 261:8708–8711PubMedGoogle Scholar
  11. 11.
    van der Sluijs P, Hull M, Webster P, Male P, Goud B, Mellman I (1992) The small GTP-binding protein rab4 controls an early sorting event on the endocytic pathway. Cell 70:729–740CrossRefPubMedGoogle Scholar
  12. 12.
    Reiterer V, Grossniklaus L, Tschon T, Kasper CA, Sorg I, Arrieumerlou C (2011) Shigella flexneri type III secreted effector OspF reveals new crosstalks of proinflammatory signaling pathways during bacterial infection. Cell Signal 23:1188–1196CrossRefPubMedGoogle Scholar
  13. 13.
    Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J et al (2006) Cell Profiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7:R100CrossRefPubMedCentralPubMedGoogle Scholar
  14. 14.
    Kriz A, Schmid K, Baumgartner N, Ziegler U, Berger I, Ballmer-Hofer K, Berger P (2010) A plasmid-based multigene expression system for mammalian cells. Nat Commun 1:120CrossRefPubMedGoogle Scholar
  15. 15.
    Macia E, Ehrlich M, Massol R, Boucrot E, Brunner C, Kirchhausen T (2006) Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 10:839–850CrossRefPubMedGoogle Scholar
  16. 16.
    Stein BS, Bensch KG, Sussman HH (1984) Complete inhibition of transferrin recycling by monensin in K562 cells. J Biol Chem 259:14762–14772PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • David T. Hirschmann
    • 1
  • Christoph A. Kasper
    • 1
  • Martin Spiess
    • 1
  1. 1.BiozentrumUniversity of BaselBaselSwitzerland

Personalised recommendations