Synapto-pHluorin is a genetically encoded optical indicator of vesicle release and recycling. It is used in neuroscience to study transmitter release. It consists of a pH-sensitive form of green fluorescent protein (GFP) fused to the luminal side of a vesicle-associated membrane protein (VAMP). At the acidic pH inside transmitter vesicles, synapto-pHluorin is non-fluorescent (quenched). When vesicles get released, synapto-pHluorin is exposed to the neutral extracellular space and the presynaptic terminal becomes brightly fluorescent. Following endocytosis, vesicles become re-acidified and the cycle can start again. Chemical alkalinization of all vesicles is often used for normalization of the synapto-pHluorin signals. Synapto-pHluorin sometimes consists of yellow fluorescent protein (YFP) to monitor the cytoplasm because its pKa is higher than GFP (7.1 versus 6.0).[1]

History

Synapto-pHluorin was invented by Gero Miesenböck in 1998.[2] In 2006, an improved version was published, using synaptophysin to target the GFP to vesicles.[3] In 2013, a two-color release sensor (ratio-sypHy) was introduced to determine the size of the recycling pool at individual synapses.[4]

Applications

Synapto-pHluorin is mainly used by neurobiologists to study transmitter release and recycling at presynaptic terminals.[4] It has also been applied to the study of insulin secretion in beta cells of the pancreas.[5]

References

  1. ^ Ashby, Michael C.; Ibaraki, Kyoko; Henley, Jeremy M. (May 2004). "It's green outside: tracking cell surface proteins with pH-sensitive GFP". Trends in Neurosciences. 27 (5): 257–261. doi:10.1016/j.tins.2004.03.010. PMID 15111007. S2CID 23124974.
  2. ^ Miesenböck, Gero; De Angelis, Dino A.; Rothman, James E. (July 1998). "Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins". Nature. 394 (6689): 192–195. Bibcode:1998Natur.394..192M. doi:10.1038/28190. ISSN 1476-4687. PMID 9671304. S2CID 4320849.
  3. ^ Granseth, Björn; Odermatt, Benjamin; Royle, Stephen J.; Lagnado, Leon (September 2006). "Clathrin-Mediated Endocytosis Is the Dominant Mechanism of Vesicle Retrieval at Hippocampal Synapses". Neuron. 51 (6): 773–786. doi:10.1016/j.neuron.2006.08.029. PMID 16982422.
  4. ^ a b Rose, Tobias; Schoenenberger, Philipp; Jezek, Karel; Oertner, Thomas G. (2013). "Developmental Refinement of Vesicle Cycling at Schaffer Collateral Synapses". Neuron. 77 (6): 1109–1121. doi:10.1016/j.neuron.2013.01.021. PMID 23522046.
  5. ^ Tsuboi, Takashi; Rutter, Guy A. (April 2003). "Multiple Forms of "Kiss-and-Run" Exocytosis Revealed by Evanescent Wave Microscopy". Current Biology. 13 (7): 563–567. doi:10.1016/S0960-9822(03)00176-3. PMID 12676086.
Categories