Journal of Fluorescence

, Volume 20, Issue 1, pp 401–405 | Cite as

Direct Visualization of Vesicle Maturation and Plasma Membrane Protein Trafficking

Short Communication

Abstract

Internalization and intracellular trafficking of membrane proteins are now recognized as essential mechanisms that contribute to a number of cellular processes. Current methods lack the ability to specifically label the plasma membrane of a live cell, follow internalization of labeled membrane molecules, and conclusively differentiate newly formed membrane-derived vesicles from pre-existing endocytic or secretory structures in the cytoplasm. Here, we detail a visualization method for surface biotinylation of plasma membrane-derived vesicles that allows us to follow their progress from membrane to cytosol at specific time points. Using the transmembrane receptor RET as a model, we demonstrate how this method can be applied to identify plasma membrane-derived vesicle maturation, determine RET’s presence within these structures, and monitor RET’s recycling to the cell surface. This method improves on static and less discriminatory methods, providing a tool for analysis of real-time vesicle trafficking that is applicable to many systems.

Keywords

Membrane protein Internalization Recycling Biotinylation RET 

Supplementary material

10895_2009_548_MOESM1_ESM.doc (42 kb)
Table 1Data used to define theoretical point spread functions for deconvolution. (DOC 42 kb)

References

  1. 1.
    Gould GW, Lippincott-Schwartz J (2009) New roles for endosomes: from vesicular carriers to multi-purpose platforms. Nat Rev Mol Cell Biol 10(4):287–292CrossRefPubMedGoogle Scholar
  2. 2.
    Sorkin A, Von Zastrow M (2002) Signal transduction and endocytosis: close encounters of many kinds. Nat Rev Mol Cell Biol 3(8):600–614CrossRefPubMedGoogle Scholar
  3. 3.
    Winterstein C, Trotter J, Kramer-Albers EM (2008) Distinct endocytic recycling of myelin proteins promotes oligodendroglial membrane remodeling. J Cell Sci 121(Pt 6):834–842CrossRefPubMedGoogle Scholar
  4. 4.
    Richardson DS, Lai AZ, Mulligan LM (2006) RET ligand-induced internalization and its consequences for downstream signaling. Oncogene 25(22):3206–3211CrossRefPubMedGoogle Scholar
  5. 5.
    Richardson DS, Gujral TS, Peng S et al (2009) Transcript level modulates the inherent oncogenicity of RET/PTC oncoproteins. Cancer Res 69(11):4861–4869CrossRefPubMedGoogle Scholar
  6. 6.
    Gujral TS, Singh VK, Jia Z et al (2006) Molecular mechanisms of RET receptor-mediated oncogenesis in multiple endocrine neoplasia 2B. Cancer Res 66(22):10741–10749CrossRefPubMedGoogle Scholar
  7. 7.
    Smith CA, Dho SE, Donaldson J, Tepass U, McGlade CJ (2004) The cell fate determinant numb interacts with EHD/Rme-1 family proteins and has a role in endocytic recycling. Mol Biol Cell 15(8):3698–3708CrossRefPubMedGoogle Scholar
  8. 8.
    Gujral TS, van Veelen W, Richardson DS et al (2008) A novel RET kinase-beta-catenin signaling pathway contributes to tumorigenesis in thyroid carcinoma. Cancer Res 68(5):1338–1346CrossRefPubMedGoogle Scholar
  9. 9.
    Shroff H, Galbraith CG, Galbraith JA et al (2007) Dual-color superresolution imaging of genetically expressed probes within individual adhesion complexes. Proc Natl Acad Sci U S A 104(51):20308–20313CrossRefPubMedGoogle Scholar
  10. 10.
    Abramoff MD, Magelhaes PJ, Ram SJ (2004) Image processing with ImageJ. Biophoton Int 11(7):36–42Google Scholar
  11. 11.
    French AR, Sudlow GP, Wiley HS et al (1994) Postendocytic trafficking of epidermal growth factor-receptor complexes is mediated through saturable and specific endosomal interactions. J Biol Chem 269(22):15749–15755PubMedGoogle Scholar
  12. 12.
    Herbst JJ, Opresko LK, Walsh BJ et al (1994) Regulation of postendocytic trafficking of the epidermal growth factor receptor through endosomal retention. J Biol Chem 269(17):12865–12873PubMedGoogle Scholar
  13. 13.
    Marshall S, Green A, Olefsky JM (1981) Evidence for recycling of insulin receptors in isolated rat adipocytes. J Biol Chem 256(22):11464–11470PubMedGoogle Scholar
  14. 14.
    Grampp T, Notz V, Broll I et al (2008) Constitutive, agonist-accelerated, recycling and lysosomal degradation of GABA(B) receptors in cortical neurons. Mol Cell Neurosci 39(4):628–637CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Department of Pathology and Molecular Medicine, Division of Cancer Biology and Genetics, Cancer Research InstituteQueen’s UniversityKingstonCanada

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