Skip to main content

Spatiotemporal Analysis of Caveolae Dynamics Using Total Internal Reflection Fluorescence Microscopy

  • Protocol
  • First Online:
Caveolae

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2169))

Abstract

Total internal reflection fluorescence microscopy enables to analyze the localizations and dynamics of cellular events that occur at or near the plasma membrane. Total internal reflection fluorescence microscopy exclusively illuminates molecules in the close vicinity of the glass surface, thereby reducing background fluorescence and enabling observation of the plasma membrane in the glass-attached cells with a high signal-to-noise ratio. Here, we describe the application of total internal reflection fluorescence microscopy to analyze the dynamics of caveolae, which play essential physiological functions, including membrane tension buffering, endocytosis, and signaling at the plasma membrane.

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 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
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. Sako Y, Minoghchi S, Yanagida T (2000) Single-molecule imaging of EGFR signalling on the surface of living cells. Nat Cell Biol 2:168–172

    Article  CAS  Google Scholar 

  2. Boucrot E, Howes MT, Kirchhausen T, Parton RG (2011) Redistribution of caveolae during mitosis. J Cell Sci 124:1965–1972

    Article  CAS  Google Scholar 

  3. Johnson A, Vert G (2017) Single event resolution of plant plasma membrane protein endocytosis by TIRF microscopy. Front Plant Sci 8:612

    Article  Google Scholar 

  4. Mattheyses AL, Simon SM, Rappoport JZ (2010) Imaging with total internal reflection fluorescence microscopy for the cell biologist. J Cell Sci 123:3621–3628

    Article  CAS  Google Scholar 

  5. Rosendale M, Perrais D (2017) Imaging in focus: imaging the dynamics of endocytosis. Int J Biochem Cell Biol 93:41–45

    Article  CAS  Google Scholar 

  6. Senju Y, Itoh Y, Takano K et al (2011) Essential role of PACSIN2/syndapin-II in caveolae membrane sculpting. J Cell Sci 124:2032–2040

    Article  CAS  Google Scholar 

  7. Senju Y, Suetsugu S (2015) Possible regulation of caveolar endocytosis and flattening by phosphorylation of F-BAR domain protein PACSIN2/Syndapin II. BioArchitecture 5:70–77

    Article  Google Scholar 

  8. Sinha B, Köster D, Ruez R et al (2011) Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144:402–413

    Article  CAS  Google Scholar 

  9. Senju Y, Rosenbaum E, Shah C et al (2015) Phosphorylation of PACSIN2 by protein kinase C triggers the removal of caveolae from the plasma membrane. J Cell Sci 128:2766–2780

    Article  CAS  Google Scholar 

  10. Tachikawa M, Morone N, Senju Y et al (2017) Measurement of caveolin-1 densities in the cell membrane for quantification of caveolar deformation after exposure to hypotonic membrane tension. Sci Rep 7:7794

    Article  Google Scholar 

  11. Lamaze C, Tardif N, Dewulf M et al (2017) The caveolae dress code: structure and signaling. Curr Opin Cell Biol 47:117–125

    Article  CAS  Google Scholar 

  12. Grassart A, Cheng AT, Hong SH et al (2014) Actin and dynamin2 dynamics and interplay during clathrin-mediated endocytosis. J Cell Biol 205:721–735

    Article  CAS  Google Scholar 

  13. Shvets E, Bitsikas V, Howard G et al (2015) Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids. Nat Commun 6:6867

    Article  CAS  Google Scholar 

  14. Macia E, Ehrlich M, Massol R et al (2006) Dynasore, a cell-permeable inhibitor of dynamin. Dev Cell 10:839–850

    Article  CAS  Google Scholar 

  15. Pédelacq J-D, Cabantous S, Tran T et al (2006) Engineering and characterization of a superfolder green fluorescent protein. Nat Biotechnol 24:79–88

    Article  Google Scholar 

  16. Yoshiyuki Arai. Fast single molecule particle tracking and analysis plugin with Java Native Interface (ImageJ User and Developer Conference). http://imagejconf.tudor.lu/program/poster/yoshiyuki_arai73473173

  17. Smith MB, Karatekin E, Gohlke A et al (2011) Interactive, computer-assisted tracking of speckle trajectories in fluorescence microscopy: application to actin polymerization and membrane fusion. Biophys J 101:1794–1804

    Article  CAS  Google Scholar 

  18. Jaqaman K, Loerke D, Mettlen M et al (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5:695–702

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the Funding Program for Next Generation World Leading Researchers (NEXT program) (grant number: LS031); Japan Society for the Promotion of Science (JSPS) KAKENHI (grant numbers: 26291037, 15H01641, 15H05902). Astellas Foundation for Research on Metabolic Disorders to S.S.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Yosuke Senju or Shiro Suetsugu .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Senju, Y., Suetsugu, S. (2020). Spatiotemporal Analysis of Caveolae Dynamics Using Total Internal Reflection Fluorescence Microscopy. In: Blouin, C. (eds) Caveolae. Methods in Molecular Biology, vol 2169. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0732-9_6

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0732-9_6

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0731-2

  • Online ISBN: 978-1-0716-0732-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics