Caveolae pp 71-80 | Cite as

Live-Cell FRET Imaging of Phosphorylation-Dependent Caveolin-1 Switch

  • Adriana M. Zimnicka
  • Zhenlong Chen
  • Peter T. Toth
  • Richard D. MinshallEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2169)


The detection of dynamic conformational changes in proteins in live cells is challenging. Live-cell FRET (Förster Resonance Energy Transfer) is an example of a noninvasive technique that can be used to achieve this goal at nanometer resolution. FRET-based assays are dependent on the presence of fluorescent probes, such as CFP- and YFP-conjugated protein pairs. Here, we describe an experimental protocol in which live-cell FRET was used to measure conformational changes in caveolin-1 (Cav-1) oligomers on the surface of plasmalemma vesicles, or caveolae.

Key words

Live-cell FRET Cav-1-CFP Cav-1-YFP Caveolae Conformation change 


  1. 1.
    Fernandez I, Ying Y, Albanesi J, Anderson RG (2002) Mechanism of caveolin filament assembly. Proc Natl Acad Sci U S A 99:11193–11198CrossRefGoogle Scholar
  2. 2.
    Shvets E, Ludwig A, Nichols BJ (2014) News from the caves: update on the structure and function of caveolae. Curr Opin Cell Biol 29:99–106CrossRefGoogle Scholar
  3. 3.
    Parton RG, Simons K (2007) The multiple faces of caveolae. Nat Rev Mol Cell Biol 8:185–194CrossRefGoogle Scholar
  4. 4.
    Sverdlov M, Shinin V, Place AT, Castellon M, Minshall RD (2009) Filamin A regulates caveolae internalization and trafficking in endothelial cells. Mol Biol Cell 20:4531–4540CrossRefGoogle Scholar
  5. 5.
    Lisanti MP, Tang Z, Scherer PE, Kubler E, Koleske AJ, Sargiacomo M (1995) Caveolae, transmembrane signalling and cellular transformation. Mol Membr Biol 12:121–124CrossRefGoogle Scholar
  6. 6.
    Fielding CJ, Fielding PE (2001) Caveolae and intracellular trafficking of cholesterol. Adv Drug Deliv Rev 49:251–264CrossRefGoogle Scholar
  7. 7.
    Sinha B, Koster D, Ruez R, Gonnord P, Bastiani M, Abankwa D, Stan RV, Butler-Browne G, Vedie B, Johannes L, Morone N, Parton RG, Raposo G, Sens P, Lamaze C, Nassoy P (2011) Cells respond to mechanical stress by rapid disassembly of caveolae. Cell 144:402–413CrossRefGoogle Scholar
  8. 8.
    Zimnicka AM, Husain YS, Shajahan AN, Sverdlov M, Chaga O, Chen Z, Toth PT, Klomp J, Karginov AV, Tiruppathi C, Malik AB, Minshall RD (2016) Src-dependent phosphorylation of caveolin-1 Tyr-14 promotes swelling and release of caveolae. Mol Biol Cell 27:2090–2106CrossRefGoogle Scholar
  9. 9.
    Shimomura O, Johnson FH, Saiga Y (1962) Extraction, purification and properties of aequorin, a bioluminescent protein from the luminous hydromedusan, Aequorea. J Cell Comp Physiol 59:223–239CrossRefGoogle Scholar
  10. 10.
    Shrestha D, Jenei A, Nagy P, Vereb G, Szollosi J (2015) Understanding FRET as a research tool for cellular studies. Int J Mol Sci 16:6718–6756CrossRefGoogle Scholar
  11. 11.
    Gauer JW, LeBlanc S, Hao P, Qiu R, Case BC, Sakato M, Hingorani MM, Erie DA, Weninger KR (2016) Single-molecule FRET to measure conformational dynamics of DNA mismatch repair proteins. Methods Enzymol 581:285–315CrossRefGoogle Scholar
  12. 12.
    Katz RA, Merkel G, Andrake MD, Roder H, Skalka AM (2011) Retroviral integrases promote fraying of viral DNA ends. J Biol Chem 286:25710–25718CrossRefGoogle Scholar
  13. 13.
    Ben-Johny M, Yue DN, Yue DT (2016) Detecting stoichiometry of macromolecular complexes in live cells using FRET. Nat Commun 7:13709CrossRefGoogle Scholar
  14. 14.
    Butz ES, Ben-Johny M, Shen M, Yang PS, Sang L, Biel M, Yue DT, Wahl-Schott C (2016) Quantifying macromolecular interactions in living cells using FRET two-hybrid assays. Nat Protoc 11:2470–2498CrossRefGoogle Scholar
  15. 15.
    Chen Z, Bakhshi FR, Shajahan AN, Sharma T, Mao M, Trane A, Bernatchez P, van Nieuw Amerongen GP, Bonini MG, Skidgel RA, Malik AB, Minshall RD (2012) Nitric oxide-dependent Src activation and resultant caveolin-1 phosphorylation promote eNOS/caveolin-1 binding and eNOS inhibition. Mol Biol Cell 23:1388–1398CrossRefGoogle Scholar
  16. 16.
    Snapp EL, Hegde RS, Francolini M, Lombardo F, Colombo S, Pedrazzini E, Borgese N, Lippincott-Schwartz J (2003) Formation of stacked ER cisternae by low affinity protein interactions. J Cell Biol 163:257–269CrossRefGoogle Scholar
  17. 17.
    Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544CrossRefGoogle Scholar
  18. 18.
    Matz MV, Fradkov AF, Labas YA, Savitsky AP, Zaraisky AG, Markelov ML, Lukyanov SA (1999) Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol 17:969–973CrossRefGoogle Scholar
  19. 19.
    von Stetten D, Noirclerc-Savoye M, Goedhart J, Gadella TW Jr, Royant A (2012) Structure of a fluorescent protein from Aequorea victoria bearing the obligate-monomer mutation A206K. Acta Crystallogr Sect F Struct Biol Cryst Commun 68:878–882CrossRefGoogle Scholar
  20. 20.
    Zacharias DA, Violin JD, Newton AC, Tsien RY (2002) Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296:913–916CrossRefGoogle Scholar
  21. 21.
    Ouyang M, Sun J, Chien S, Wang Y (2008) Determination of hierarchical relationship of Src and Rac at subcellular locations with FRET biosensors. Proc Natl Acad Sci 105:14353–14358CrossRefGoogle Scholar
  22. 22.
    Nguyen AW, Daugherty PS (2005) Evolutionary optimization of fluorescent proteins for intracellular FRET. Nat Biotechnol 23:355–360CrossRefGoogle Scholar
  23. 23.
    Shaner NC, Steinbach PA, Tsien RY (2005) A guide to choosing fluorescent proteins. Nat Methods 2:905–909CrossRefGoogle Scholar
  24. 24.
    Elder AD, Domin A, Kaminski Schierle GS, Lindon C, Pines J, Esposito A, Kaminski CF (2009) A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission. J R Soc Interface 6:S59–S81CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Adriana M. Zimnicka
    • 1
  • Zhenlong Chen
    • 2
  • Peter T. Toth
    • 1
    • 3
  • Richard D. Minshall
    • 1
    • 2
    Email author
  1. 1.Department of PharmacologyUniversity of Illinois at ChicagoChicagoUSA
  2. 2.Department of and AnesthesiologyUniversity of Illinois at ChicagoChicagoUSA
  3. 3.Research Resources Center Fluorescence Imaging CoreUniversity of Illinois at ChicagoChicagoUSA

Personalised recommendations