Skip to main content
SpringerLink
Log in

Nanoclustering and Heterogeneous Membrane Diffusion of Ras Studied by FRAP and RICS Analysis

  • Protocol
  • First Online:
Ras Signaling

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1120))

Abstract

Fluorescence Recovery After Photobleaching (FRAP) and Raster Image Correlation Spectroscopy (RICS) are two powerful techniques to study the diffusion dynamics of fluorescently labeled proteins. FRAP and RICS can be easily applied on any commercial confocal microscope. In this chapter, we describe the principles of these methods and provide the reader with a detailed guide on how to apply these methods in the study of Ras nanoclustering and diffusion in the plasma membrane of live cells.

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

Access this chapter

Log in via an institution
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. Abankwa D, Gorfe AA, Hancock JF (2007) Ras nanoclusters: molecular structure and assembly. Semin Cell Dev Biol 18:599–607

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  2. Prior IA, Muncke C, Parton RG et al (2003) Direct visualization of Ras proteins in spatially distinct cell surface microdomains. J Cell Biol 160:165–170

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. Plowman SJ, Muncke C, Parton RG et al (2005) H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton. Proc Natl Acad Sci U S A 102: 15500–15505

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  4. Hancock JF, Parton RG (2005) Ras plasma membrane signalling platforms. Biochem J 389:1–11

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  5. Janosi L, Li Z, Hancock JF et al (2012) Organization, dynamics, and segregation of Ras nanoclusters in membrane domains. Proc Natl Acad Sci U S A 109:8097–8102

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  6. Belanis L, Plowman SJ, Rotblat B et al (2008) Galectin-1 is a novel structural component and a major regulator of h-ras nanoclusters. Mol Biol Cell 19:1404–1414

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  7. Rotblat B, Belanis L, Liang H et al (2010) H-Ras nanocluster stability regulates the magnitude of MAPK signal output. PLoS One. doi:10.1371/journal.pone.0011991

    Google Scholar 

  8. Shalom-Feuerstein R, Plowman SJ, Rotblat B et al (2008) K-ras nanoclustering is subverted by overexpression of the scaffold protein galectin-3. Cancer Res 68:6608–6616

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  9. Matsunaga-Udagawa R, Fujita Y, Yoshiki S et al (2010) The scaffold protein Shoc2/SUR-8 accelerates the interaction of Ras and Raf. J Biol Chem 285:7818–7826

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  10. Inder KL, Lau C, Loo D et al (2009) Nucleophosmin and nucleolin regulate K-Ras plasma membrane interactions and MAPK signal transduction. J Biol Chem 284:28410–28419

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  11. Zhou Y, Cho K-J, Plowman SJ et al (2012) Nonsteroidal anti-inflammatory drugs alter the spatiotemporal organization of Ras proteins on the plasma membrane. J Biol Chem 287:16586–16595

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  12. Köhnke M, Schmitt S, Ariotti N et al (2012) Design and application of in vivo FRET biosensors to identify protein prenylation and nanoclustering inhibitors. Chem Biol 19:866–874

    Article  PubMed  Google Scholar 

  13. Paz A, Haklai R, Elad-Sfadia G et al (2001) Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation. Oncogene 20:7486–7493

    Article  PubMed  CAS  Google Scholar 

  14. Tian T, Harding A, Inder K et al (2007) Plasma membrane nanoswitches generate high-fidelity Ras signal transduction. Nat Cell Biol 9:905–914

    Article  PubMed  CAS  Google Scholar 

  15. Lommerse PHM, Snaar-Jagalska BE, Spaink HP et al (2005) Single-molecule diffusion measurements of H-Ras at the plasma membrane of live cells reveal microdomain localization upon activation. J Cell Sci 118: 1799–1809

    Article  PubMed  CAS  Google Scholar 

  16. Murakoshi H, Iino R, Kobayashi T et al (2004) Single-molecule imaging analysis of Ras activation in living cells. Proc Natl Acad Sci U S A 101:7317–7322

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  17. Abankwa D, Vogel H (2007) A FRET map of membrane anchors suggests distinct microdomains of heterotrimeric G proteins. J Cell Sci 120:2953–2962

    Article  PubMed  CAS  Google Scholar 

  18. Abankwa D, Hanzal-Bayer M, Ariotti N et al (2008) A novel switch region regulates H-ras membrane orientation and signal output. EMBO J 27:727–735

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  19. Axelrod D, Koppel DE, Schlessinger J et al (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys J 16:1055–1069

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  20. Feder TJ, Brust-Mascher I, Slattery JP et al (1996) Constrained diffusion or immobile fraction on cell surfaces: a new interpretation. Biophys J 70:2767–2773

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  21. Perez J-B, Segura JM, Abankwa D et al (2006) Monitoring the diffusion of single heterotrimeric G proteins in supported cell-membrane sheets reveals their partitioning into microdomains. J Mol Biol 363:918–930

    Article  PubMed  CAS  Google Scholar 

  22. Digman MA, Brown CM, Sengupta P et al (2005) Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys J 89:1317–1327

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  23. Brown CM, Dalal RB, Hebert B et al (2008) Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope. J Microsc 229:78–91

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  24. Rossow MJ, Sasaki JM, Digman MA et al (2010) Raster image correlation spectroscopy in live cells. Nat Protoc 5:1761–1774

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  25. Abankwa D, Gorfe AA, Inder K et al (2010) Ras membrane orientation and nanodomain localization generate isoform diversity. Proc Natl Acad Sci U S A 107:1130–1135

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  26. Schneider CA, Rasband WS, Eliceiri KW (2012) NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675

    Article  PubMed  CAS  Google Scholar 

  27. Kapusta P (2010) Absolute diffusion coefficients: compilation of reference data for FCS calibration. Application note: PicoQuant GmbH, Berlin. http://www.picoquant.com/images/uploads/page/files/7353/appnote_diffusioncoefficients.pdf

  28. Gielen E, Smisdom N, vandeVen M et al (2009) Measuring diffusion of lipid-like probes in artificial and natural membranes by raster image correlation spectroscopy (RICS): use of a commercial laser-scanning microscope with analog detection. Langmuir 25:5209–5218

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Buschmann V, Krämer B, Koberling F et al (2009) Quantitative FCS: determination of the confocal volume by FCS and bead scanning with the MicroTime 200. Application note: PicoQuant GmbH, Berlin. http://www.picoquant.com/images/uploads/page/files/7351/appnote_quantfcs.pdf

Download references

Author information

Authors and Affiliations

  1. Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland

    Camilo Guzmán, Maja Šolman & Daniel Abankwa

Authors
  1. Camilo Guzmán
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maja Šolman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Daniel Abankwa
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. University of Siena, Italy

    Lorenza Trabalzini

  2. University of Torino, Italy

    Saverio Francesco Retta

Rights and permissions

Reprints and permissions

Copyright information

© 2014 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Guzmán, C., Šolman, M., Abankwa, D. (2014). Nanoclustering and Heterogeneous Membrane Diffusion of Ras Studied by FRAP and RICS Analysis. In: Trabalzini, L., Retta, S. (eds) Ras Signaling. Methods in Molecular Biology, vol 1120. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-791-4_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-62703-791-4_20

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-790-7

  • Online ISBN: 978-1-62703-791-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation