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Stoichiometry of Receptors at the Plasma Membrane During Their Endocytosis Using Total Internal Reflection Fluorescent (TIRF) Microscopy Live Imaging and Single-Molecule Tracking

  • Laura Salavessa
  • Nathalie SauvonnetEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2233)

Abstract

Determination of protein stoichiometry in living cells is key to understanding basic biological processes. This is particularly important for receptor-mediated endocytosis, a highly regulated mechanism that requires the sequential assembly of numerous factors. Here, we describe a quantitative approach to analyze receptor clustering dynamics at the plasma membrane. Our workflow combines TIRF live imaging of a CRISPR-Cas9-edited cell line expressing a GFP-tagged receptor in a physiological relevant environment, a calibration technique for single-molecule analysis of GFP, and detection and tracking with an open-source software. This method allows to determine the number of receptor molecules at the plasma membrane in real time.

Key words

Single-molecule tracking (SPT) TIRF Endocytosis Photobleaching Receptor Dynamics Plasma membrane Quantitative biology 

Notes

Acknowledgments

First, we would like to thank Dr. Alexandre Grassart for training us with the technologies of CRISPR/Cas9 gene edition and single-molecule analysis and for all helpful discussion. We thank the PBI (Imagopole) platform of Institut Pasteur for microscope maintenance and technical help. LS is part of the Pasteur Paris University (PPU) International PhD Program and has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement No 665807.

References

  1. 1.
    Clayton AHA, Walker F, Orchard SG et al (2005) Ligand-induced dimer-tetramer transition during the activation of the cell surface epidermal growth factor receptor-A multidimensional microscopy analysis. J Biol Chem 280:30392–30399CrossRefGoogle Scholar
  2. 2.
    Liang SI, van Lengerich B, Eichel K et al (2018) Phosphorylated EGFR dimers are not sufficient to activate Ras. Cell Rep 22:2593–2600CrossRefGoogle Scholar
  3. 3.
    Needham SR, Roberts SK, Arkhipov A et al (2016) EGFR oligomerization organizes kinase-active dimers into competent signalling platforms. Nat Commun 7:13307CrossRefGoogle Scholar
  4. 4.
    Huang Y, Bharill S, Karandur D et al (2016) Molecular basis for multimerization in the activation of the epidermal growth factor receptor. Elife 5:e14107CrossRefGoogle Scholar
  5. 5.
    Nakano Y, Monde K, Terasawa H et al (2014) Preferential recognition of monomeric CCR5 expressed in cultured cells by the HIV-1 envelope glycoprotein gp120 for the entry of R5 HIV-1. Virology 452–453:117–124CrossRefGoogle Scholar
  6. 6.
    Colin P, Zhou Z, Staropoli I et al (2018) CCR5 structural plasticity shapes HIV-1 phenotypic properties. PLoS Pathog 14:e1007432CrossRefGoogle Scholar
  7. 7.
    Vila-Coro AJ, Mellado M, Martín de Ana A et al (2000) HIV-1 infection through the CCR5 receptor is blocked by receptor dimerization. Proc Natl Acad Sci U S A 97:3388–3393CrossRefGoogle Scholar
  8. 8.
    Kaksonen M, Roux A (2018) Mechanisms of clathrin-mediated endocytosis. Nat Rev Mol Cell Biol 19:313–326CrossRefGoogle Scholar
  9. 9.
    Cocucci E, Aguet F, Boulant S et al (2012) The first five seconds in the life of a Clathrin-coated pit. Cell 150:495–507CrossRefGoogle Scholar
  10. 10.
    Grassart A, Cheng AT, Hong SH et al (2014) Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis. J Cell Biol 205:721–735CrossRefGoogle Scholar
  11. 11.
    Cocucci E, Gaudin R, Kirchhausen T (2014) Dynamin recruitment and membrane scission at the neck of a clathrin-coated pit. Mol Biol Cell 25:3595–3609CrossRefGoogle Scholar
  12. 12.
    Lamaze C, Dujeancourt A, Baba T et al (2001) Interleukin 2 receptors and detergent-resistant membrane domains define a Clathrin-independent endocytic pathway. Mol Cell 7:661–671CrossRefGoogle Scholar
  13. 13.
    Gesbert F, Sauvonnet N, Dautry-Varsat A (2004) Clathrin-lndependent endocytosis and signalling of interleukin 2 receptors IL-2R endocytosis and signalling. Curr Top Microbiol Immunol 286:119–148PubMedGoogle Scholar
  14. 14.
    Basquin C, Trichet M, Vihinen H et al (2015) Membrane protrusion powers clathrin-independent endocytosis of interleukin-2 receptor. EMBO J:1–15Google Scholar
  15. 15.
    Hori T, Uchiyama T, Tsudo M et al (1987) Establishment of an interleukin 2-dependent human T cell line from a patient with T cell chronic lymphocytic leukemia who is not infected with human T cell leukemia/lymphoma virus. Blood 70:1069–1072CrossRefGoogle Scholar
  16. 16.
    de Chaumont F, Dallongeville S, Chenouard N et al (2012) Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods 9:690–696CrossRefGoogle Scholar
  17. 17.
    Bertot L, Grassart A, Lagache T et al (2018) Quantitative and statistical study of the dynamics of Clathrin-dependent and -independent endocytosis reveal a differential role of EndophilinA2. Cell Rep 22:1574–1588CrossRefGoogle Scholar
  18. 18.
    Yildiz A, Vale RD (2015) Total internal reflection fluorescence microscopy. Cold Spring Harb Protoc 2015:pdb.top086348CrossRefGoogle Scholar
  19. 19.
    Chen Y, Deffenbaugh NC, Anderson CT et al (2014) Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex. Mol Biol Cell 25:3630–3642CrossRefGoogle Scholar
  20. 20.
    Tsekouras K, Custer TC, Jashnsaz H et al (2016) A novel method to accurately locate and count large numbers of steps by photobleaching. Mol Biol Cell 27:3601–3615CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  1. 1.Group intracellular trafficking and tissue homeostasis. Unité de Pathogénie Microbienne MoléculaireInstitut PasteurParisFrance
  2. 2.U1202, INSERMParisFrance
  3. 3.Université Paris Sud, Paris-Saclay UniversityOrsayFrance

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