Photoactivation and Imaging of Photoactivatable Fluorescent Proteins

George H. Patterson1

1 Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 21.6
DOI:  10.1002/0471143030.cb2106s38
Online Posting Date:  March, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

A major advance in the microscopic study of cells and tissues is the introduction of photoactivatable fluorescent proteins, which can specifically mark proteins of interest within a living cell. Fluorescent proteins are now available that allow a pool of molecules to be “turned on” by photoactivation. This unit discusses technical aspects for the general use of photoactivatable fluorescent proteins and introduces some specific applications in the concluding remarks. Curr. Protoc. Cell Biol. 38:21.6.1‐21.6.10. © 2008 by John Wiley & Sons, Inc.

Keywords: photoactivatable; fluorescent protein; microscopy

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Background
  • Requirements for Photoactivating Fluorescent Proteins
  • Optimization Procedures
  • General Photoactivation Experiment
  • Uses of Photoactivatable Fluorescent Proteins
  • Future Directions of PA‐FPs
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Ando, R., Hama, H., Yamamoto‐Hino, M., Mizuno, H., and Miyawaki, A. 2002. An optical marker based on the UV‐induced green‐to‐red photoconversion of a fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 99: 12651‐12656.
   Ando, R., Mizuno, H., and Miyawaki, A. 2004. Regulated fast nucleocytoplasmic shuttling observed by reversible protein highlighting. Science 306: 1370‐1373.
   Betzig, E., Patterson, G.H., Sougrat, R., Lindwasser, O.W., Olenych, S., Bonifacino, J.S., Davidson, M.W., Lippincott‐Schwartz, J., and Hess, H.F. 2006. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313: 1642‐1645.
   Campbell, R.E., Tour, O., Palmer, A.E., Steinbach, P.A., Baird, G.S., Zacharias, D.A., and Tsien, R.Y. 2002. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 99: 7877‐7882.
   Cheezum, M.K., Walker, W.F., and Guilford, W.H. 2001. Quantitative comparison of algorithms for tracking single fluorescent particles. Biophys. J. 81: 2378‐2388.
   Chudakov, D.M., Belousov, V.V., Zaraisky, A.G., Novoselov, V.V., Staroverov, D.B., Zorov, D.B., Lukyanov, S., and Lukyanov, K.A. 2003. Kindling fluorescent proteins for precise in vivo photolabeling. Nat. Biotechnol. 21: 191‐194.
   Chudakov, D.M., Verkhusha, V.V., Staroverov, D.B., Souslova, E.A., Lukyanov, S., and Lukyanov, K.A. 2004. Photoswitchable cyan fluorescent protein for protein tracking. Nat. Biotechnol. 22: 1435‐1439.
   Day, R.N., Periasamy, A., and Schaufele, F. 2001. Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus. Methods 25: 4‐18.
   Demarco, I.A., Periasamy, A., Booker, C.F., and Day, R.N. 2006. Monitoring dynamic protein interactions with photoquenching FRET. Nat. Methods 3: 519‐524.
   Ehrig, T., O'Kane, D.J., and Prendergast, F.G. 1995. Green‐fluorescent protein mutants with altered fluorescence excitation spectra. FEBS Lett. 367: 163‐166.
   Gurskaya, N.G., Verkhusha,V.V., Shcheglov, A.S., Staroverov, D.B., Chepurnykh, T.V., Fradkov, A.F., Lukyanov, S., and Lukyanov, K.A. 2006. Engineering of a monomeric green‐to‐red photoactivatable fluorescent protein induced by blue light. Nat. Biotechnol. 24: 461‐465.
   Heim, R., Prasher, D.C., and Tsien, R.Y. 1994. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. U.S.A. 91: 12501‐12504.
   Hofmann, M., Eggeling, C., Jakobs, S., and Hell, S.W. 2005. Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. Proc. Natl. Acad. Sci. U.S.A. 102: 17565‐17569.
   Kim, P.K., Mullen, R.T., Schumann, U., and Lippincott‐Schwartz, J. 2006. The origin and maintenance of mammalian peroxisomes involves a de novo PEX16‐dependent pathway from the ER. J. Cell Biol. 173: 521‐532.
   Lukyanov, K.A., Fradkov, A.F., Gurskaya, N.G., Matz, M.V., Labas, Y.A., Savitsky, A.P., Markelov, M.L., Zaraisky, A.G., Zhao, X., Fang, Y., Tan, W., and Lukyanov, S.A. 2000. Natural animal coloration can be determined by a nonfluorescent green fluorescent protein homolog. J. Biol. Chem. 275: 25879‐25882.
   Lukyanov, K.A., Chudakov, D.M., Lukyanov, S., and Verkhusha, V.V. 2005. Innovation: Photoactivatable fluorescent proteins. Nat. Rev. Mol. Cell Biol. 6: 885‐891.
   Patterson, G.H. and Lippincott‐Schwartz, J. 2002. A photoactivatable GFP for selective photolabeling of proteins and cells. Science 297: 1873‐1877.
   Post, J.N., Lidke, K.A., Rieger, B., and Arndt‐Jovin, D.J. 2005. One‐ and two‐photon photoactivation of a paGFP‐fusion protein in live Drosophila embryos. FEBS Lett. 579: 325‐330.
   Schneider, M., Barozzi, S., Testa, I., Faretta, M., and Diaspro, A. 2005. Two‐photon activation and excitation properties of PA‐GFP in the 720‐920‐nm region. Biophys. J. 89: 1346‐1352.
   Thompson, R.E., Larson, D.R., and Webb, W.W. 2002. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82: 2775‐2783.
   Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N., and Miyawaki, A. 2005. Semi‐rational engineering of a coral fluorescent protein into an efficient highlighter. EMBO Rep. 6: 233‐238.
   Verkhusha, V.V. and Sorkin, A. 2005. Conversion of the monomeric red fluorescent protein into a photoactivatable probe. Chem. Biol. 12: 279‐285.
   Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K.D., and Nienhaus, G.U. 2004. EosFP, a fluorescent marker protein with UV‐inducible green‐to‐red fluorescence conversion. Proc. Natl. Acad. Sci. U.S A. 101: 15905‐15910.
   Yokoe, H. and Meyer, T. 1996. Spatial dynamics of GFP‐tagged proteins investigated by local fluorescence enhancement. Nat. Biotechnol. 14: 1252‐1256.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library