Skip to main content
SpringerLink
Log in

Total Internal Reflection Fluorescence Microscopy to Study GLUT4 Trafficking

  • Protocol
  • First Online:
Glucose Transport

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1713))

Abstract

Total internal reflection fluorescence (TIRF) microscopy is a powerful method that allows examination of plasma membrane close events in real time. The last decade, the method has successfully been used to explore GLUT4 translocation in adipocytes. Here, we describe the procedure for studying GLUT4 trafficking using TIRF microscopy in isolated primary adipocytes.

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

Access this chapter

Log in via an institution

Institutional subscriptions

References

  1. Bryant NJ, Govers R, James DE (2002) Regulated transport of the glucose transporter GLUT4. Nat Rev Mol Cell Biol 3(4):267–277. https://doi.org/10.1038/nrm782

    Article  CAS  PubMed  Google Scholar 

  2. Stenkula KG, Lizunov VA, Cushman SW, Zimmerberg J (2010) Insulin controls the spatial distribution of GLUT4 on the cell surface through regulation of its postfusion dispersal. Cell Metab 12(3):250–259. https://doi.org/10.1016/j.cmet.2010.08.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Lizunov VA, Lisinski I, Stenkula K, Zimmerberg J, Cushman SW (2009) Insulin regulates fusion of GLUT4 vesicles independent of Exo70-mediated tethering. J Biol Chem 284(12):7914–7919. https://doi.org/10.1074/jbc.M806460200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lizunov VA, Matsumoto H, Zimmerberg J, Cushman SW, Frolov VA (2005) Insulin stimulates the halting, tethering, and fusion of mobile GLUT4 vesicles in rat adipose cells. J Cell Biol 169(3):481–489. https://doi.org/10.1083/jcb.200412069

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Lizunov VA, Stenkula K, Troy A, Cushman SW, Zimmerberg J (2013) Insulin regulates Glut4 confinement in plasma membrane clusters in adipose cells. PLoS One 8(3):e57559. https://doi.org/10.1371/journal.pone.0057559

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Bai L, Wang Y, Fan J, Chen Y, Ji W, Qu A, Xu P, James DE, Xu T (2007) Dissecting multiple steps of GLUT4 trafficking and identifying the sites of insulin action. Cell Metab 5(1):47–57. https://doi.org/10.1016/j.cmet.2006.11.013

    Article  CAS  PubMed  Google Scholar 

  7. Huang S, Lifshitz LM, Jones C, Bellve KD, Standley C, Fonseca S, Corvera S, Fogarty KE, Czech MP (2007) Insulin stimulates membrane fusion and GLUT4 accumulation in clathrin coats on adipocyte plasma membranes. Mol Cell Biol 27(9):3456–3469. https://doi.org/10.1128/MCB.01719-06

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Xu Y, Rubin BR, Orme CM, Karpikov A, Yu C, Bogan JS, Toomre DK (2011) Dual-mode of insulin action controls GLUT4 vesicle exocytosis. J Cell Biol 193(4):643–653. https://doi.org/10.1083/jcb.201008135

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Jiang L, Fan J, Bai L, Wang Y, Chen Y, Yang L, Chen L, Xu T (2008) Direct quantification of fusion rate reveals a distal role for AS160 in insulin-stimulated fusion of GLUT4 storage vesicles. J Biol Chem 283(13):8508–8516. https://doi.org/10.1074/jbc.M708688200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Lizunov VA, Stenkula KG, Blank PS, Troy A, Lee JP, Skarulis MC, Cushman SW, Zimmerberg J (2015) Human adipose cells in vitro are either refractory or responsive to insulin, reflecting host metabolic state. PLoS One 10(3):e0119291. https://doi.org/10.1371/journal.pone.0119291

    Article  PubMed  PubMed Central  Google Scholar 

  11. Lizunov VA, Lee JP, Skarulis MC, Zimmerberg J, Cushman SW, Stenkula KG (2013) Impaired tethering and fusion of GLUT4 vesicles in insulin-resistant human adipose cells. Diabetes 62(9):3114–3119. https://doi.org/10.2337/db12-1741

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Poulter NS, Pitkeathly WT, Smith PJ, Rappoport JZ (2015) The physical basis of total internal reflection fluorescence (TIRF) microscopy and its cellular applications. Methods Mol Biol 1251:1–23. https://doi.org/10.1007/978-1-4939-2080-8_1

    Article  CAS  PubMed  Google Scholar 

  13. Rodbell M (1964) Metabolism of isolated fat cells. I. Effects of hormones on glucose metabolism and lipolysis. J Biol Chem 239:375–380

    CAS  PubMed  Google Scholar 

  14. Stenkula KG, Said L, Karlsson M, Thorn H, Kjolhede P, Gustavsson J, Soderstrom M, Stralfors P, Nystrom FH (2004) Expression of a mutant IRS inhibits metabolic and mitogenic signalling of insulin in human adipocytes. Mol Cell Endocrinol 221(1–2):1–8. https://doi.org/10.1016/j.mce.2004.04.011

    Article  CAS  PubMed  Google Scholar 

  15. Burchfield JG, Lu J, Fazakerley DJ, Tan SX, Ng Y, Mele K, Buckley MJ, Han W, Hughes WE, James DE (2013) Novel systems for dynamically assessing insulin action in live cells reveals heterogeneity in the insulin response. Traffic 14(3):259–273. https://doi.org/10.1111/tra.12035

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Experimental Medical Science, Lund University, BMC C11, Lund, 22 184, Sweden

    Sebastian Wasserstrom, Björn Morén & Karin G. Stenkula

Authors
  1. Sebastian Wasserstrom
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Björn Morén
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Karin G. Stenkula
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Karin G. Stenkula .

Editor information

Editors and Affiliations

  1. Department of Experimental Medical Science, Lund University, Lund, Sweden

    Karin Lindkvist-Petersson

  2. Department of Experimental Medical Science, Lund University, Lund, Sweden

    Jesper S Hansen

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Science+Business Media LLC

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Wasserstrom, S., Morén, B., Stenkula, K.G. (2018). Total Internal Reflection Fluorescence Microscopy to Study GLUT4 Trafficking. In: Lindkvist-Petersson, K., Hansen, J. (eds) Glucose Transport. Methods in Molecular Biology, vol 1713. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7507-5_12

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-7507-5_12

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7506-8

  • Online ISBN: 978-1-4939-7507-5

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation