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Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins


In neural systems, information is often carried by ensembles of cells rather than by individual units. Optical indicators1 provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA2,3,4: encodable probes can be introduced into cells, tissues, or transgenic organisms by genetic manipulation, selectively expressed in anatomically or functionally defined groups of cells, and, ideally, recorded in situ, without a requirement for exogenous cofactors. Here we describe sensors for secretion and neurotransmission that fulfil these criteria. We have developed pH-sensitive mutants of green fluorescent protein (‘pHluorins’) by structure-directed combinatorial mutagenesis, with the aim of exploiting the acidic pH inside secretory vesicles5,6 to monitor vesicle exocytosis and recycling. When linked to a vesicle membrane protein, pHluorins were sorted to secretory and synaptic vesicles and reported transmission at individual synaptic boutons, as well as secretion and fusion pore ‘flicker’ of single secretory granules.

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Figure 1: Fluorescence excitation spectra.
Figure 2: pH measurements with ratiometric pHluorin in HeLa cells.
Figure 3: Neurotransmission visualized with ratiometric pHluorin.
Figure 4: Secretion visualized with ecliptic pHluorin.


  1. Tsien, R. Y. Fluorescent probes of cell signaling. Annu. Rev. Neurosci. 12, 227–253 (1989).

    Article  CAS  Google Scholar 

  2. Miesenböck, G. & Rothman, J. E. Patterns of synaptic activity in neural networks recorded by light emission from synaptolucins. Proc. Natl Acad. Sci. USA 94, 3402–3407 (1997).

    Article  ADS  Google Scholar 

  3. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882–887 (1997).

    Article  ADS  CAS  Google Scholar 

  4. Romoser, V. A., Hinkle, P. M. & Persechini, A. Detection in living cells of Ca2+-dependent changes in the fluorescence emission of an indicator composed of two green fluorescent protein variants linked by a calmodulin-binding sequence. J. Biol. Chem. 272, 13270–13274 (1997).

    Article  CAS  Google Scholar 

  5. Anderson, R. G. & Orci, L. Aview of acidic intracellular compartments. J. Cell Biol. 106, 539–543 (1988).

    Article  CAS  Google Scholar 

  6. Südhof, T. C. The synaptic vesicle cycle: a cascade of protein–protein interactions. Nature 375, 645–653 (1995).

    Article  ADS  Google Scholar 

  7. Ward, W. W. in Bioluminescence and Chemiluminescence (eds DeLuca, M. A. & McElroy, W. D.) 235–242 (Academic, New York, (1981)).

    Book  Google Scholar 

  8. Ward, W. W., Prentice, H. J., Roth, A. F., Cody, C. W. & Reeves, S. C. Spectral perturbations of the Aequorea green-fluorescent protein. Photochem. Photobiol. 35, 803–808 (1982).

    Article  CAS  Google Scholar 

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

    Article  ADS  CAS  Google Scholar 

  10. Chattoraj, M., King, B. A., Bublitz, G. U. & Boxer, S. G. Ultra-fast excited state dynamics in green fluorescent protein: Multiple states and proton transfer. Proc. Natl Acad. Sci. USA 93, 8362–8367 (1996).

    Article  ADS  CAS  Google Scholar 

  11. Ormö, M. et al. Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395 (1996).

    Article  ADS  Google Scholar 

  12. Yang, F., Moss, L. G. & Phillips, G. N. J The molecular structure of green fluorescent protein. Nature Biotechnol. 14, 1246–1251 (1996).

    Article  CAS  Google Scholar 

  13. Brejc, K. et al. Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc. Natl Acad. Sci. USA 94, 2306–2311 (1997).

    Article  ADS  CAS  Google Scholar 

  14. Ehrig, T., O'Kane, D. J. & Prendergast, F. G. Green-fluorescent protein mutants with altered fluorescence excitation spectra. FEBS Lett. 367, 163–166 (1995).

    Article  CAS  Google Scholar 

  15. Heim, R. & Tsien, R. Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182 (1996).

    Article  CAS  Google Scholar 

  16. Caras, I. W., Weddell, G. N., Davitz, M. A., Nussenzweig, V. & Martin, D. W. J Signal for attachment of a phospholipid membrane anchor in decay accelerating factor. Science 238, 1280–1283 (1987).

    Article  ADS  CAS  Google Scholar 

  17. Luzio, J. P. et al. Identification, sequencing and expression of an integral membrane protein of the trans-Golgi network (TGN38). Biochem. J. 270, 97–102 (1990).

    Article  CAS  Google Scholar 

  18. McMahon, H. T. et al. Cellubrevin is a ubiquitous tetanus-toxin substrate homologous to a putative synaptic vesicle fusion protein. Nature 364, 346–349 (1993).

    Article  ADS  CAS  Google Scholar 

  19. Marconi, P. et al. Replication-defective herpes simplex virus vectors for gene transfer in vivo. Proc. Natl Acad. Sci. USA 93, 11319–11320 (1996).

    Article  ADS  CAS  Google Scholar 

  20. Lawrence, M. S., Ho, D. Y., Dash, R. & Sapolsky, R. M. Herpes simplex virus vectors overexpressing the glucose transporter gene protect against seizure-induced neuron loss. Proc. Natl Acad. Sci. USA 92, 7247–7251 (1995).

    Article  ADS  CAS  Google Scholar 

  21. Stevens, C. F. & Tsujimoto, T. Estimates for the pool size of releasable quanta at a single central synapse and for the time required to refill the pool. Proc. Natl Acad. Sci. USA 92, 846–849 (1995).

    Article  ADS  CAS  Google Scholar 

  22. Roa, M., Paumet, F., Le Mao, J., David, B. & Blank, U. Involvement of the ras-like GTPase rab3d inRBL-2H3 mast cell exocytosis following stimulation via high-affinity IgE receptors (FcεRI). J.Immunol. 159, 2815–2823 (1997).

    PubMed  CAS  Google Scholar 

  23. Fernandez, J. M., Neher, E. & Gomperts, B. D. Capacitance measurements reveal stepwise fusion events in degranulating mast cells. Nature 312, 453–455 (1984).

    Article  ADS  CAS  Google Scholar 

  24. Chandler, D. E. & Heuser, J. E. Arrest of membrane fusion events in mast cells by quick-freezing. J. Cell Biol. 86, 666–674 (1980).

    Article  CAS  Google Scholar 

  25. Ullrich, A. & Schlessinger, J. Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203–212 (1990).

    Article  CAS  Google Scholar 

  26. Yu, S. S., Lefkowitz, R. J. & Hausdorff, W. P. β-Adrenergic receptor sequestration: A potential mechanism of receptor resensitization. J. Biol. Chem. 268, 337–341 (1993).

    PubMed  CAS  Google Scholar 

  27. James, D. E. & Piper, R. C. Insulin resistance, diabetes, and the insulin-regulated trafficking of GLUT-4. J. Cell Biol. 126, 1123–1126 (1994).

    Article  CAS  Google Scholar 

  28. Siemering, K. R., Golbik, R., Sever, R. & Haseloff, J. Mutations that suppress the thermosensitivity of green fluorescent protein. Curr. Biol. 6, 1653–1663 (1996).

    Article  CAS  Google Scholar 

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We thank R. Miller for technical assistance, D. Krisky and J. Glorioso for HSV strain THZ.3 and 7B cells, G. Schiavo for BoNT, the Fonds de la Recherche en Santé du Québec for a postdoctoral fellowship (to D.A.D.), and Q. Al-Awqati for discussion. This research was supported by the G. Harold and Leila Y. Mathers Charitable Foundation.

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Miesenböck, G., De Angelis, D. & Rothman, J. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

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