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Fluorescence Imaging with Single-Molecule Sensitivity and Fluorescence Correlation Spectroscopy of Cell-Penetrating Neuropeptides

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Neuropeptides

Part of the book series: Methods in Molecular Biology ((MIMB,volume 789))

Abstract

Neuropeptide–plasma membrane interactions in the absence of a corresponding specific receptor may result in neuropeptide translocation into the cell. Translocation across the plasma membrane may represent a previously unknown mechanism by which neuropeptides can signal information to the cell interior. We introduce here two complementary optical methods with single-molecule sensitivity, fluorescence imaging with avalanche photodiode detectors (APD imaging) and fluorescence correlation spectroscopy (FCS), and demonstrate how they may be applied for the analysis of neuropeptide ability to penetrate into live cells in real time. APD imaging enables us to visualize fluorescently labeled neuropeptide molecules at very low, physiologically relevant concentrations, whereas FCS enables us to characterize quantitatively their concentration and diffusion properties in different cellular compartments. Application of these methodologies for the analysis of the endogenous opioid peptide dynorphin A (Dyn A), a ligand for the kappa-opioid receptor (KOP), demonstrated that this neuropeptide may translocate across the plasma membrane of living cells and enter the cellular interior without binding to its cognate receptor.

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References

  1. Strand, F. L. (1999) Neuropeptides: Regulators of Physiological Processes, Publisher: Massachusetts Institute of Technology.

    Google Scholar 

  2. Revest, P., and Longstaff, A. (1998) Molecular Neuroscience, Publisher: BIOS Scientific Publishers Limited.

    Google Scholar 

  3. Lorenz, D., Wiesner, B., Zipper, J., Winkler, A., Krause, E., Beyermann, M., Lindau, M., and Bienert, M. (1998) Mechanism of peptide-induced mast cell degranulation. Translocation and patch-clamp studies. J. Gen. Physiol. 112, 577–591.

    Article  PubMed  CAS  Google Scholar 

  4. Marinova, Z., Vukojević, V., Surcheva, S., Yakovleva, T., Cebers, G., Pasikova, N., Usynin, I., Hugonin, L., Fang, W., Hallberg, M., Hirschberg, D., Bergman, T., Langel, U., Hauser, K. F., Pramanik, A., Aldrich, J. V., Gräslund, A., Terenius, L., and Bakalkin G. (2005) Translocation of dynorphin neuropeptides across the plasma membrane. A putative mechanism of signal transmission. J. Biol. Chem. 280, 26360–26370.

    Article  PubMed  CAS  Google Scholar 

  5. Saban, R., Gerard, N. P., Saban, M. R., Nguyen, N. B., DeBoer, D. J., and Wershil, B. K. (2002) Mast cells mediate substance P-induced bladder inflammation through an NK(1) receptor-independent mechanism. Am. J. Physiol. Renal Physiol. 283, F616–629.

    PubMed  Google Scholar 

  6. Hauser, K. F., Aldrich, J. V., Anderson, K. J., Bakalkin, G., Christie, M. J., Hall, E. D., Knapp, P. E., Scheff, S. W., Singh, I. N., Vissel, B., Woods, A. S., Yakovleva, T., and Shippenberg, T. S. (2005) Pathobiology of dynorphins in trauma and disease. Front. Biosci. 10, 216–235.

    Article  PubMed  CAS  Google Scholar 

  7. Tan-No, K., Takahashi, H., Nakagawasai, O., Nijima, F., Sato, T., Satoh, S., Sakurada, S, Marinova, Z., Yakovleva, T., Bakalkin, G., Terenius, L., and Tadano, T. (2005) Pronociceptive role of dynorphins in uninjured animals: N-ethylmaleimide-induced nociceptive behavior mediated through inhibition of dynorphin degradation. Pain 113, 301–309.

    Article  PubMed  CAS  Google Scholar 

  8. Kuzmin, A., Madjid, N., Terenius, L., Ogren, S. O., and Bakalkin, G. (2006) Big dynorphin, a prodynorphin-derived peptide produces NMDA receptor-mediated effects on memory, anxiolytic-like and locomotor behavior in mice. Neuropsychopharmacology 31, 1928–1937.

    Article  PubMed  CAS  Google Scholar 

  9. Woods, A. S., Kaminski, R., Oz, M., Wang, Y., Hauser, K., Goody, R., Wang, H. Y., Jackson, S. N., Zeitz, P., Zeitz, K. P., Zolkowska, D., Schepers, R., Nold, M., Danielson, J., Gräslund, A., Vukojević, V., Bakalkin, G., Basbaum, A., and Shippenberg, T. (2006) Decoy peptides that bind dynorphin noncovalently prevent NMDA receptor-mediated neurotoxicity. J. Proteome Res. 5, 1017–1023.

    Article  PubMed  CAS  Google Scholar 

  10. Wadia, J. S., Stan, R. V., and Dowdy, S. F. (2004) Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315.

    Article  PubMed  CAS  Google Scholar 

  11. Joliot, A., and Prochiantz, A. (2004) Transduction peptides: from technology to physiology. Nat. Cell Biol. 6, 189–196.

    Article  PubMed  CAS  Google Scholar 

  12. Järver, P., and Langel, Ü. (2004) The use of cell-penetrating peptides as a tool for gene regulation. Drug Discov. Today 9, 395–402.

    Article  PubMed  Google Scholar 

  13. Prochiantz, A., and Joliot, A. (2003) Can transcription factors function as cell-cell signalling molecules? Nat. Rev. Mol. Cell Biol. 4, 814–819.

    PubMed  CAS  Google Scholar 

  14. Vukojević, V., Heidkamp, M., Ming, Y., Johansson, B., Terenius, L., and Rigler, R. (2008) Quantitative single-molecule imaging by Confocal Laser Scanning Microscopy. Proc. Natl. Acad. Sci. USA 105, 18176–18181.

    Article  PubMed  Google Scholar 

  15. Elson, E. L. (2001) Fluorescence correlation spectroscopy measures molecular transport in cells. Traffic 2, 789–796.

    Article  PubMed  CAS  Google Scholar 

  16. Bacia, K., and Schwille, P. (2003) A dynamic view of cellular processes by in vivo fluorescence auto- and cross-correlation spectroscopy. Methods 29, 74–85.

    Article  PubMed  CAS  Google Scholar 

  17. Elson, E. L. (2004) Quick tour of fluorescence correlation spectroscopy from its inception. J. Biomed. Optics. 9, 857–864.

    Article  CAS  Google Scholar 

  18. Vukojević, V., Pramanik, A., Yakovleva, T., Rigler, R., Terenius, L., and Bakalkin, G. (2005) Study of Molecular Events in Cells by Fluorescence Correlation Spectroscopy. Cell. Mol. Life Sci. 62, 535–550.

    Article  PubMed  Google Scholar 

  19. García-Sáez, A. J., and Schwille, P. (2007) Single molecule techniques for the study of membrane proteins. Appl. Microbiol. Biotechnol. 76, 257–266.

    Article  PubMed  Google Scholar 

  20. Haustein, E., and Schwille, P. (2007) Fluorescence correlation spectroscopy: novel variations of an established technique. Annu. Rev. Biophys. Biomol. Struct. 36, 151–169.

    Article  PubMed  CAS  Google Scholar 

  21. Bacia, K., and Schwille, P. (2007) Practical guidelines for dual-color fluorescence cross-correlation spectroscopy. Nat. Protoc. 2, 2842–2856.

    Article  PubMed  CAS  Google Scholar 

  22. Vukojević, V., Papadopoulos, D. K., Terenius, L., Gehring, W., and Rigler, R. (2010) Quantitative study of synthetic Hox transcription factor–DNA interactions in live cells. Proc. Natl. Acad. Sci. USA 107, 4087–4092.

    Article  PubMed  Google Scholar 

  23. Magde, D., Webb, W. W. and Elson, E. (1972) Thermodynamic fluctuations in a reacting system - measurement by fluorescence correlation spectroscopy. Phys. Rev. Lett. 29, 705–708.

    Article  CAS  Google Scholar 

  24. Elson, E. L., and Magde, D. (1974) Fluorescence correlation spectroscopy. Conceptual basis and theory. Biopolymers 13, 1–27.

    Article  CAS  Google Scholar 

  25. Ehrenberg, M., and Rigler, R. (1974) Rotational Brownian motion and fluorescence intensity fluctuations. Chem. Phys. 4, 390–401.

    Article  CAS  Google Scholar 

  26. Koppel, D. E. (1974) Statistical accuracy in fluorescence correlation spectroscopy. Phys. Rev. A 10, 1938–1945.

    Article  Google Scholar 

  27. Koppel, D. E., Axelrod, D., Schlessinger, J., Elson, E. L., and Webb, W. W. (1976). Dynamics of fluorescence marker concentration as a probe of mobility. Biophys. J. 16, 1315–1329.

    Article  PubMed  CAS  Google Scholar 

  28. Axelrod, D., Koppel, D. E., Schlessinger, J., Elson, E., and Webb, W. W. (1976) Mobility measurement by analysis of fluorescence photobleaching recovery kinetics. Biophys. J. 16, 1055–1069.

    Article  PubMed  CAS  Google Scholar 

  29. Elson, E. L., Schlessinger, J., Koppel, D. E., Axelrod, D., and Webb, W. W. (1976) Measurement of lateral transport on cell surfaces. Prog. Clin. Biol. Res. 9, 137–147.

    PubMed  CAS  Google Scholar 

  30. Schlessinger, J., Koppel, D. E., Axelrod, D., Jacobson, K., Webb, W. W., and Elson, E. L. (1976) Lateral transport on cell membranes: Mobility of concanavalin A receptors on myoblasts. Proc. Natl. Acad. Sci. USA 73, 2409–2413.

    Google Scholar 

  31. Schlessinger, J., Axelrod, D., Koppel, D. E., Webb, W. W., and Elson, E. L. (1977) Lateral transport of a lipid probe and labeled proteins on a cell membrane. Science 195, 307–309.

    Article  PubMed  CAS  Google Scholar 

  32. Jacobson, K., Elson, E., Koppel, D., and Webb, W. W. (1982) Fluorescence photobleaching in cell biology. Nature 295, 283–284.

    Article  PubMed  CAS  Google Scholar 

  33. Yechiel, E., and Edidin, M. (1987) Micrometer-scale domains in fibroblast plasma membranes. J. Cell Biol. 105, 755–760.

    Article  PubMed  CAS  Google Scholar 

  34. Berland, K. M., So, P. T. C., and Gratton, E. (1995) Two-photon fluorescence correlation spectroscopy: Method and application to the intracellular environment. Biophys. J. 68, 694–701.

    Article  PubMed  CAS  Google Scholar 

  35. Schwille, P., Haupts, U., Maiti, S., and Webb, W. W. (1999) Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys. J. 77, 2251–2265.

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  37. Zipfel, W. R., Williams, R. M., Christie, A., Nikitin, A.Y., Hyman, B. T., and Webb, W. W. (2003) Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. USA 100, 7075–7080.

    Article  PubMed  CAS  Google Scholar 

  38. Zipfel, W. R., Williams, R. M., and Webb, W. W. (2003) Nonlinear Magic: Multiphoton microscopy in the biosciences. Nature Biotech. 21, 1369–1377.

    Article  CAS  Google Scholar 

  39. Miyawaki, A., Sawano, A., and Kogure, T. (2003) Lighting up cells: labelling proteins with fluorophores. Nat. Cell Biol. Suppl: S1–S7.

    Google Scholar 

  40. Machleidt, T., Robers, M., and Hanson, G. T. (2007) Protein labeling with FlAsH and ReAsH. Methods Mol Biol. 356, 209–220.

    PubMed  CAS  Google Scholar 

  41. Thompson, N. L. (1991) Fluorescence correlation spectroscopy. In: Topics in fluorescence Spectroscopy, Volume 1: Techniques, pp 337–378, Lakowicz J. R. (ed.), Plenum Press, New York.

    Google Scholar 

  42. Qian, H., and Elson, E. L. (1990) On the analysis of high order moments of fluorescence fluctuations. Biophys. J. 57, 375–380.

    Article  PubMed  CAS  Google Scholar 

  43. Kask, P., Palo, K., Ullmann, D., and Gall, K. (1999) Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc. Natl. Acad. Sci. USA 96, 13756–13761.

    Article  PubMed  CAS  Google Scholar 

  44. Kask, P., Palo, K., Fay, N., Brand, L., Mets, U., Ullmann, D. et al. (2000) Two-dimensional fluorescence intensity distribution analysis: theory and applications. Biophys. J. 78, 1703–1713.

    Article  PubMed  CAS  Google Scholar 

  45. Chen, Y., Müller, J. D., So, P. T. C., and Gratton, E. (1999) The photon counting ­histogram in fluorescence fluctuation spectroscopy. Biophys. J. 77, 553–567.

    Article  PubMed  CAS  Google Scholar 

  46. Hillesheim, L. N., and Müller, J. D. (2003) The photon counting histogram in fluorescence fluctuation spectroscopy with non-ideal photodetectors. Biophys. J. 85, 1948–1958.

    Article  PubMed  CAS  Google Scholar 

  47. Müller, J. D. (2004) Cumulant analysis in fluorescence fluctuation spectroscopy. Biophys. J. 86, 3981–3992.

    Article  PubMed  Google Scholar 

  48. Weisshart, K., Jüngel, V., and Briddon, S. J. (2004) The LSM 510 META - ConfoCor 2 system: an integrated imaging and spectroscopic platform for single-molecule detection. Curr. Pharm. Biotechnol. 5, 135–154.

    Article  PubMed  CAS  Google Scholar 

  49. Müller, C.B., Loman, A., Pacheco, V., Koberling, F., Willbold, D., Richtering, W., and Enderlein, J. (2008) Precise measurement of diffusion by multi-color dual-focus fluorescence correlation spectroscopy, EPL-Europhys. Lett. 83, 46001.

    Article  Google Scholar 

  50. Giner, D., Ñeco, P., del Mar Francés, M., López, I., Viniegra, S., and Gutiérrez, L. M. (2005) Real-time dynamics of the F-actin cytoskeleton during secretion from chromaffin cells. J. Cell. Sci. 118, 2871–2880.

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We thank Carl-Jonas Lindskog for testing the protocol and procedures described in this work. VV acknowledges support from the Knut and Alice Wallenberg Foundation, Swedish Research Council, Swedish Brain Foundation, Ministry of Sciences and Technological Development of Serbia (Grants no. 142025 and 142019). This work was also supported by grants from the Swedish Council for Working Life and Social Research (FAS) and Swedish Science Research Council to GB.

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Authors and Affiliations

  1. Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden

    Vladana Vukojević

  2. Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

    Astrid Gräslund

  3. Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden

    Georgy Bakalkin

Authors
  1. Vladana Vukojević
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  2. Astrid Gräslund
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  3. Georgy Bakalkin
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Correspondence to Georgy Bakalkin .

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Editors and Affiliations

  1. , Dipt. di Morfofisiologia Veterinaria, Università degli Studi di Torino, Via Leonardo da Vinci 44, Grugliasco, 10095, Torino, Italy

    Adalberto Merighi

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Vukojević, V., Gräslund, A., Bakalkin, G. (2011). Fluorescence Imaging with Single-Molecule Sensitivity and Fluorescence Correlation Spectroscopy of Cell-Penetrating Neuropeptides. In: Merighi, A. (eds) Neuropeptides. Methods in Molecular Biology, vol 789. Humana Press. https://doi.org/10.1007/978-1-61779-310-3_9

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  • DOI: https://doi.org/10.1007/978-1-61779-310-3_9

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