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Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement

Nature Biotechnology volume 14, pages 1252–1256 (1996)Cite this article

Abstract

We describe a method of monitoring the spatial dynamics of proteins in intact cells by locally enhancing the blue excited fluorescence of green fluorescent protein (GFP) using a spatially focused ultraviolet-laser pulse. GFP fusion proteins were efficiently expressed by micro-electroporation of in vitro synthesized mRNA into adherent mammalian cells. We found that the diffusion coefficient of cycle 3 mutant GFP was 43 μm2/sec, compared to 4 μm2/sec for wild-type GFP, suggesting that cycle 3 GFP diffuses freely in mammalian cells and is ideally suited as a fusion tag. The local fluorescence enhancement method was used to study the membrane dissociation rate of GFP-tagged K-ras, a small GTP binding protein that localizes to plasma membranes by a farnesyl lipid group and a polybasic region. Our data suggest that K-ras exists in a dynamic equilibrium and rapidly switches between a plasma membrane bound form and a cytosolic form with a plasma membrane dissociation time constant of 1.5 sec.

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References

  1. Schlessinger, J. and Bar-Sagi, D. 1994. Activation of Ras and other signaling pathways by receptor tyrosine kinases. Cold Spring Harbor Symposia on Quantitative Biology 59: 173–179.

    Article  CAS  Google Scholar 

  2. Coghlan, V.M., Perrino, B.A., Howard, M., Langeberg, L.K., Hicks, J.B., Gallatin, W.M. et al. 1995. Association of protein kinase A and protein phosphatase 2B with a common anchoring protein. Science 287: 108–111.

    Article  Google Scholar 

  3. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., and Prasher, D.C. 1994. Green fluorescent protein as a marker for gene expression. Science 263: 802–805.

    Article  CAS  Google Scholar 

  4. Prasher, D.G., Eckenrode, V.K., Ward, W.W., Prendergast, F.G. and Cormier, M.J. 1992. Primary structure of the Aequorea victoria green fluorescent protein. Gene 111: 229–233.

    Article  CAS  Google Scholar 

  5. Heim, R. and Tsien, R.Y. 1996. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence energy transfer. Current Biology 6: 178–182

    Article  CAS  Google Scholar 

  6. Stearns, T. 1995. The green revolution. Current Biology 5: 3.

    Article  Google Scholar 

  7. Crameri, A., Whitehorn, E.A., Tate, E. and Stemmer, W.P.C. 1996. Improved green fluorescent protein by molecular evolution using DNA shuffling. Nature Biotechnology 14: 315–319.

    Article  CAS  Google Scholar 

  8. Falcone, D. and Andrews, D.W. 1991. Both 5′ untranslated region and the sequences surrounding the start site contribute to efficient initiation of translation in vitro . Mol. and Cell. Biol. 11: 2565–2664.

    Google Scholar 

  9. Green, M.R., Maniatis, T. and Melton, D.A. 1983. Human beta-globin pre-mRNA synthesized in vitro is accurately spliced in Xenopus oocyte nuclei. Cell 32: 681–694.

    Article  CAS  Google Scholar 

  10. Kozak, M. 1987. An analysis of 5′-noncoding sequences from 699 vertebrate messenger RNAs. Nucl. Acid Res. 15: 8125–8148.

    Article  CAS  Google Scholar 

  11. Tanguay, R.L. and Gallie, D.R. 1996. Translational efficiency is regulated by the length of the 3′ untranslated region. Mol. Cell. Biol. 16: 146–156.

    Article  CAS  Google Scholar 

  12. Heim, R., Prasher, D.C. and Tsien, R.Y. 1994. Wavelength mutations and posttranslational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA 91: 12501–12504.

    Article  CAS  Google Scholar 

  13. Cubitt, A.B., Heim, R., Adams, S.R., Boyd, A.E., Gross, L.A., and Tsien, R.Y. 1995. Understanding, improving and using green fluorescent proteins. TIBS 20: 448–455.

    CAS  PubMed  Google Scholar 

  14. Mastro, A.M. and Keith, A.D. 1984. Diffusion in the aqueous compartment. J. Cell Biol. 99: 180s–187s.

    Article  CAS  Google Scholar 

  15. Luby-Phelps, K., Mujumdar, S., Mujumdar, R.B., Ernst, L.A., Galbraith, W., and Waggoner, A.S. 1994. A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm. Biophys. J. 65: 236–242.

    Article  Google Scholar 

  16. Boguski, M.S. and McCormick, F. 1993. Proteins regulating Ras and its relatives. Nature 366: 643–654.

    Article  CAS  Google Scholar 

  17. Casey, P.J. 1995. Protein lipidation in cell signaling. Science 268: 221–225.

    Article  CAS  Google Scholar 

  18. Le Gal La Salle, G., Robert, J.J., Berrard, S., Ridoux, V., Stratford-Perricaudet, L.D., Perricaudet, M. et al. 1993. An adenovirus vector for gene transfer into neurons and glia in the brain. Science 259: 988–990.

    Article  CAS  Google Scholar 

  19. Claudio, T. 1992. Stable expression of heterologous multisubunit protein complexes established by calcium phosphate- or lipid-mediated cotransfection. Methods Enzymol. 207: 391–408.

    Article  CAS  Google Scholar 

  20. Herskowitz, I. 1987. Functional inactivation of genes by dominant negative mutations. Nature 329: 219–222.

    Article  CAS  Google Scholar 

  21. Nghiem, P., Ollick, T., Gardner, P., and Schulman, H. 1994. lnterleukin-2 transcriptional block by multifunctional Ca2+/calmodulin kinase. Nature 371: 347–350.

    Article  CAS  Google Scholar 

  22. Zhou, Q.Y., Grandy, O.K., Thambi, L., Kushner, J.A., Van Tol, H.H., Cone, R., et al. 1990. Cloning and expression of human and rat D1 dopamine receptors. Nature 347: 76–80.

    Article  CAS  Google Scholar 

  23. Mitchison, T.J. and Salmon, E.D. 1992. Poleward kinetochore fiber movement occurs during both metaphase and anaphase-A in newt lung cell mitosis. J. Cell Biol. 119: 569–582.

    Article  CAS  Google Scholar 

  24. Denk, W., Strickler, J.H. and Webb, W.W. 1990. Two-photon laser scanning fluorescence microscopy. Science 248: 73–76.

    Article  CAS  Google Scholar 

  25. Ausubel, F.M. 1996. Current Protocols in Molecular Biology, chapter 3.9.1. John Wiley and Sons, New York.

    Google Scholar 

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  1. Tobias Meyer: email: tobias@cellbio.duke.edu

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  1. Department of Cell Biology, Duke University Medical Center, Durham, NC, 27710

    Hiroko Yokoe & Tobias Meyer

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  1. Hiroko Yokoe
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  2. Tobias Meyer
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Yokoe, H., Meyer, T. Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Nat Biotechnol 14, 1252–1256 (1996). https://doi.org/10.1038/nbt1096-1252

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