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 indicators provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA: 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 vesicles 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.
Miesenböck
G
G
Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
De Angelis
D A
DA
Rothman
J E
JE
D000818:Animals; D002478:Cells, Cultured; D003594:Cytoplasmic Granules / Q000502:physiology; D005089:Exocytosis; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D011993:Recombinant Fusion Proteins; D009435:Synaptic Transmission; D013572:Synaptic Vesicles / Q000502:physiology
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C514578:synaptopHIuorin; D049452:Green Fluorescent Proteins