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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.


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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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