Brain Cell Biology

, Volume 36, Issue 1–4, pp 31–42 | Cite as

Highly sensitive and quantitative FRET–FLIM imaging in single dendritic spines using improved non-radiative YFP

Article

Abstract

Two-photon fluorescence lifetime imaging microscopy (TPFLIM) enables the quantitative measurements of fluorescence resonance energy transfer (FRET) in small subcellular compartments in light scattering tissue. We evaluated and optimized the FRET pair of mEGFP (monomeric EGFP with the A206K mutation) and REACh (non-radiative YFP variants) for TPFLIM. We characterized several mutants of REACh in terms of their “darkness,” and their ability to act as a FRET acceptor for mEGFP in HeLa cells and hippocampal neurons. Since the commonly used monomeric mutation A206K increases the brightness of REACh, we introduced a different monomeric mutation (F223R) which does not affect the brightness. Also, we found that the folding efficiency of original REACh, as measured by the fluorescence lifetime of a mEGFP–REACh tandem dimer, was low and variable from cell to cell. Introducing two folding mutations (F46L, Q69M) into REACh increased the folding efficiency by ∼50%, and reduced the variability of FRET signal. Pairing mEGFP with the new REACh (super-REACh, or sREACh) improved the signal-to-noise ratio compared to the mEGFP–mRFP or mEGFP–original REACh pair by ∼50%. Using this new pair, we demonstrated that the fraction of actin monomers in filamentous and globular forms in single dendritic spines can be quantitatively measured with high sensitivity. Thus, the mEGFP–sREACh pair is suited for quantitative FRET measurement by TPFLIM, and enables us to measure protein–protein interactions in individual dendritic spines in brain slices with high sensitivity.

References

  1. Alvarez, V. A., Sabatini, B. L. (2007). Anatomical and physiological plasticity of dendritic spines. Annu. Rev. Neurosci. 30, 79–97PubMedCrossRefGoogle Scholar
  2. Bassell, G. J., Zhang, H., Byrd, A. L., Femino, A. M., Singer, R. H., Taneja, K. L., Lifshitz, L. M., Herman, I. M., Kosik, K. S. (1998). Sorting of beta-actin mRNA and protein to neurites and growth cones in culture. J. Neurosci. 18, 251–265PubMedGoogle Scholar
  3. Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A., Tsien, R. Y. (2002). A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA 99, 7877–7882PubMedCrossRefGoogle Scholar
  4. Eom, T., Antar, L. N., Singer, R. H., Bassell, G. J. (2003). Localization of a beta-actin messenger ribonucleoprotein complex with zipcode-binding protein modulates the density of dendritic filopodia and filopodial synapses. J. Neurosci. 23, 10433–10444PubMedGoogle Scholar
  5. Fischer, A., Sananbenesi, F., Schrick, C., Spiess, J., Radulovic, J. (2004). Distinct roles of hippocampal de novo protein synthesis and actin rearrangement in extinction of contextual fear. J. Neurosci. 24, 1962–1966PubMedCrossRefGoogle Scholar
  6. Ganesan, S., Ameer-Beg, S. M., Ng, T. T., Vojnovic, B., Wouters, F. S. (2006). A dark yellow fluorescent protein (YFP)-based Resonance Energy-Accepting Chromoprotein (REACh) for Forster resonance energy transfer with GFP. Proc. Natl. Acad. Sci. USA 103, 4089–4094PubMedCrossRefGoogle Scholar
  7. Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A., Tsien, R. Y. (2001). Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194PubMedCrossRefGoogle Scholar
  8. Hering, H., Sheng, M. (2003). Activity-dependent redistribution and essential role of cortactin in dendritic spine morphogenesis. J. Neurosci. 23, 11759–11769PubMedGoogle Scholar
  9. Holmes, K. C., Popp, D., Gebhard, W., Kabsch, W. (1990). Atomic model of the actin filament. Nature 347, 44–49PubMedCrossRefGoogle Scholar
  10. Honkura, N., Matsuzaki, M., Noguchi, J., Ellis-Davies, G. C., Kasai, H. (2008). The subspine organization of actin fibers regulates the structure and plasticity of dendritic spines. Neuron 57, 719–729PubMedCrossRefGoogle Scholar
  11. Kennedy, M. B., Beale, H. C., Carlisle, H. J., Washburn, L. R. (2005). Integration of biochemical signalling in spines. Nat. Rev. Neurosci. 6, 423–34PubMedCrossRefGoogle Scholar
  12. Kogure, T., Karasawa, S., Araki, T., Saito, K., Kinjo, M., Miyawaki, A. (2006). A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy. Nat. Biotechnol. 24, 577–581PubMedCrossRefGoogle Scholar
  13. Krucker, T., Siggins, G. R., Halpain, S. (2000). Dynamic actin filaments are required for stable long-term potentiation (LTP) in area CA1 of the hippocampus. Proc. Natl. Acad. Sci. USA 97, 6856–6861PubMedCrossRefGoogle Scholar
  14. Kwok, S., Lee, C., Sanchez, S. A., Hazlett, T. L., Gratton, E., Hayashi, Y. (2008). Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity. Biochem. Biophys. Res. Commun. 369, 519–525PubMedCrossRefGoogle Scholar
  15. Lakowicz, J. R. (2006). Principles of Fluorescence Spectroscopy. Plenum, NY, USAGoogle Scholar
  16. Lamprecht, R., LeDoux, J. (2004). Structural plasticity and memory. Nat. Rev. Neurosci. 5, 45–54PubMedCrossRefGoogle Scholar
  17. Matus, A. (2000). Actin-based plasticity in dendritic spines. Science 290, 754–758PubMedCrossRefGoogle Scholar
  18. McAllister, A. K. (2000). Biolistic transfection of neurons. Sci. STKE, PL1Google Scholar
  19. Miyawaki, A. (2003). Visualization of the spatial and temporal dynamics of intracellular signaling. Dev. Cell. 4, 295–305PubMedCrossRefGoogle Scholar
  20. Nagai, T., Ibata, K., Park, E. S., Kubota, M., Mikoshiba, K., Miyawaki, A. (2002). A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nat. Biotechnol. 20, 87–90PubMedCrossRefGoogle Scholar
  21. Okamoto, K., Nagai, T., Miyawaki, A., Hayashi, Y. (2004). Rapid and persistent modulation of actin dynamics regulates postsynaptic reorganization underlying bidirectional plasticity. Nat. Neurosci. 7, 1104–1112PubMedCrossRefGoogle Scholar
  22. Peter, M., Ameer-Beg, S. M., Hughes, M. K., Keppler, M. D., Prag, S., Marsh, M., Vojnovic, B., Ng, T. (2005). Multiphoton-FLIM quantification of the EGFP–mRFP1 FRET pair for localization of membrane receptor-kinase interactions. Biophys. J. 88, 1224–1237PubMedCrossRefGoogle Scholar
  23. Sekino, Y., Kojima, N., Shirao, T. (2007). Role of actin cytoskeleton in dendritic spine morphogenesis. Neurochem. Int. 51, 92–104PubMedCrossRefGoogle Scholar
  24. Shaner, N. C., Campbell, R. E., Steinbach, P. A., Giepmans, B. N., Palmer, A. E., Tsien, R. Y. (2004). Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 12, 1567–1572CrossRefGoogle Scholar
  25. Star, E. N., Kwiatkowski, D. J., Murthy, V. N. (2002). Rapid turnover of actin in dendritic spines and its regulation by activity. Nat. Neurosci. 5, 239–246PubMedCrossRefGoogle Scholar
  26. Stoppini, L., Buchs, P. A., Muller, D. A. (1991). A simple method for organotypic cultures of nervous tissue. J. Neurosci. Methods 37, 173–182PubMedCrossRefGoogle Scholar
  27. Tramier, M., Zahid, M., Mevel, J. C., Masse, M. J., Coppey-Moisan, M. (2006). Sensitivity of CFP/YFP and GFP/mCherry pairs to donor photobleaching on FRET determination by fluorescence lifetime imaging microscopy in living cells. Microsci. Res. Tech. 69, 933–939CrossRefGoogle Scholar
  28. Wang, X. B., Yang, Y., Zhou, Q. (2007). Independent expression of synaptic and morphological plasticity associated with long-term depression. J. Neurosci. 27, 12419–12429PubMedCrossRefGoogle Scholar
  29. Yasuda, R. (2006). Imaging spatiotemporal dynamics of neuronal signaling using fluorescence resonance energy transfer and fluorescence lifetime imaging microscopy. Curr. Opin. Neurobiol. 16, 551–561PubMedCrossRefGoogle Scholar
  30. Yasuda, R., Harvey, C. D., Zhong, H., Sobczyk, A., van Aelst, L., Svoboda, K. (2006). Super-sensitive Ras activation in dendrites and spines revealed by 2-photon fluorescence lifetime imaging. Nat. Neurosci. 9, 283–291PubMedCrossRefGoogle Scholar
  31. Zacharias, D. A., Violin, J. D., Newton, A. C., Tsien, R. Y. (2002). Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916PubMedCrossRefGoogle Scholar
  32. Zhang, W., Benson, D. L. (2001). Stages of synapse development defined by dependence on F-actin. J. Neurosci. 21, 5169–5181PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  1. 1.Department of NeurobiologyDuke University Medical CenterDurhamUSA

Personalised recommendations