Skip to main content

Use of Quantitative Immunofluorescence Microscopy to Study Intracellular Trafficking: Studies of the GLUT4 Glucose Transporter

  • Protocol
  • First Online:
Membrane Trafficking

Part of the book series: Methods in Molecular Biology ((MIMB,volume 457))

Summary

Insulin regulates the glucose uptake in muscle and adipose cells by acutely modulating the amount of the GLUT4 glucose transporter in the plasma membrane. The steady-state cell surface distribution of a membrane protein is an equilibrium between exocytosis and endocytosis. The authors study the effect of insulin on GLUT4 using quantitative immunofluorescence microscopy adapted to single-cell analysis. They use an HA-GLUT4-GFP reporter molecule as a surrogate of GLUT4 trafficking. Insulin induces an increase of GLUT4 in the plasma membrane by both increasing GLUT4 exocytosis and decreasing its endocytosis. Quantitative immunofluorescence techniques such as those described in this review can be used to study the trafficking of virtually any membrane protein.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 159.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Dugani C.B. and Klip A. (2005) Glucose transporter 4: cycling, compartments and controversies. EMBO Rep. 6, 1137–1142.

    Article  PubMed  CAS  Google Scholar 

  2. Ishiki M. and Klip A. (2005) Minireview: recent developments in the regulation of glucose transporter-4 traffic: new signals, locations, and partners. Endocrinology 146, 5071–5078.

    Article  PubMed  CAS  Google Scholar 

  3. Dawson K., Aviles-Hernandez A., Cushman S., and Malide D. (2001) Insulin-regulated trafficking of dual-labeled glucose transporter 4 in primary rat adipose cells. Biochem. Biophys. Res. Commun. 287, 445–454.

    Article  PubMed  CAS  Google Scholar 

  4. Lampson M.A., Racz A., Cushman S.W., McGraw T.E. (2000) Demonstration of insulin-responsive trafficking of GLUT4 and vpTR in fibroblasts. J. Cell Sci. 113, 4065–4076.

    PubMed  CAS  Google Scholar 

  5. Lampson M.A., Schmoranzer J., Zeigerer A., Simon S.M., and McGraw T.E. (2001) Insulin-regulated release from the endosomal recycling compartment is regulated by budding of specialized vesicles. Mol. Biol. Cell 12, 3489–3501.

    PubMed  CAS  Google Scholar 

  6. Zeigerer A., Lampson M., Karylowski O., et al. (2002) GLUT4 retention in adipocytes requires two intracellular insulin-regulated transport steps. Mol. Biol. Cell 13, 2421–2435.

    Article  PubMed  CAS  Google Scholar 

  7. Karylowski O., Zeigerer A., Cohen A., and McGraw T.E. (2004) GLUT4 is retained by an intracellular cycle of vesicle formation and fusion with endosomes. Mol. Biol. Cell 15, 870–882.

    Article  PubMed  CAS  Google Scholar 

  8. Dunn K., Mayor S., Myers J., and Maxfield F. (1994) Applications of ratio fluorescence microscopy in the study of cell physiology. FASEB J. 8, 573–582.

    PubMed  CAS  Google Scholar 

  9. Wiley H.S. and Cunningham D.D. (1982) The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J. Biol. Chem. 257, 4222–4229.

    PubMed  CAS  Google Scholar 

  10. Blot V. and McGraw TE. (2006) GLUT4 is internalized by a cholesterol-dependent nystatin-sensitive mechanism inhibited by insulin. Embo J. 25, 5648–5658.

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The work was supported by grants from the NIH DK52852 (TEM) and DK69982 (TEM).

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2008 Humana Press, a part of Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Blot, V., McGraw, T.E. (2008). Use of Quantitative Immunofluorescence Microscopy to Study Intracellular Trafficking: Studies of the GLUT4 Glucose Transporter . In: Vancura, A. (eds) Membrane Trafficking. Methods in Molecular Biology, vol 457. Humana Press. https://doi.org/10.1007/978-1-59745-261-8_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-261-8_26

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-925-3

  • Online ISBN: 978-1-59745-261-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics