Glutamate-sensitive fluorescent reporter

A genetically engineered fluorescent protein that changes its fluorescence when bound to the neurotransmitter glutamate. Glutamate-sensitive fluorescent reporters (iGluSnFR, colloquially pronounced 'glue sniffer') are used to monitor the activity of presynaptic terminals by fluorescence microscopy. GluSnFRs are a class of optogenetic sensors used in neuroscience research.{{Cite journal|last1=Hefendehl|first1=J. K.|last2=LeDue|first2=J.|last3=Ko|first3=R. W. Y.|last4=Mahler|first4=J.|last5=Murphy|first5=T. H.|last6=MacVicar|first6=B. A.|date=2016-11-11|title=Mapping synaptic glutamate transporter dysfunction in vivo to regions surrounding Aβ plaques by iGluSnFR two-photon imaging|journal=Nature Communications|language=En|volume=7|pages=13441|doi=10.1038/ncomms13441|pmid=27834383|bibcode=2016NatCo...713441H|pmc=5114608}} In brain tissue, two-photon microscopy is typically used to monitor GluSnFR fluorescence.

Design

File:GluSniffer Scheme.png

The widely used iGluSnFR consists of a circularly permuted enhanced green fluorescent protein (cpEGFP) fused to a glutamate binding protein (GluBP) from a bacterium.{{Cite journal|last1=Marvin|first1=Jonathan S|last2=Borghuis|first2=Bart G|last3=Tian|first3=Lin|last4=Cichon|first4=Joseph|last5=Harnett|first5=Mark T|last6=Akerboom|first6=Jasper|last7=Gordus|first7=Andrew|last8=Renninger|first8=Sabine L|last9=Chen|first9=Tsai-Wen|date=2013|title=An optimized fluorescent probe for visualizing glutamate neurotransmission|journal=Nature Methods|language=En|volume=10|issue=2|pages=162–170|doi=10.1038/nmeth.2333|pmid=23314171|issn=1548-7105|pmc=4469972}} When GluBP binds a glutamate molecule, it changes its shape, pulling the EGFP barrel together, increasing fluorescence. A specific peptide segment (PDGFR) is included to bring the sensor to the outside of the cell membrane.{{Cite journal|last1=Marvin|first1=Jonathan S.|last2=Schreiter|first2=Eric R.|last3=Echevarría|first3=Ileabett M.|last4=Looger|first4=Loren L.|date=2011-11-01|title=A genetically encoded, high-signal-to-noise maltose sensor|journal=Proteins: Structure, Function, and Bioinformatics|language=en|volume=79|issue=11|pages=3025–3036|doi=10.1002/prot.23118|pmid=21989929|issn=1097-0134|pmc=3265398}} In the more recent version by Aggarwal et al. (2022),{{Cite journal |last1=Aggarwal |first1=Abhi |last2=Liu |first2=Rui |last3=Chen |first3=Yang |last4=Ralowicz |first4=Amelia J. |last5=Bergerson |first5=Samuel J. |last6=Tomaska |first6=Filip |last7=Hanson |first7=Timothy L. |last8=Hasseman |first8=Jeremy P. |last9=Reep |first9=Daniel |last10=Tsegaye |first10=Getahun |last11=Yao |first11=Pantong |last12=Ji |first12=Xiang |last13=Kloos |first13=Marinus |last14=Walpita |first14=Deepika |last15=Patel |first15=Ronak |date=2022-02-15 |title=Glutamate indicators with improved activation kinetics and localization for imaging synaptic transmission |url=https://www.biorxiv.org/content/10.1101/2022.02.13.480251v1 |language=en |pages=2022.02.13.480251 |doi=10.1101/2022.02.13.480251|hdl=20.500.11850/613938 |hdl-access=free }} researchers introduced iGluSnFR to two additional anchoring domains, a glycosylphostidylinositol (GPI) anchor, and a modified form of the cytosolic -cterminal domain of Stargazin with a PDZ ligand.

History

The first genetically encoded fluorescent glutamate sensors (FLIPE, GluSnFR and SuperGluSnFR) were constructed by attaching cyan-fluorescent protein (CFP) and yellow-fluorescent protein (YFP) to a bacterial glutamate binding protein (GluBP).{{Cite journal|last1=Hu|first1=Yonglin|last2=Fan|first2=Cheng-Peng|last3=Fu|first3=Guangsen|last4=Zhu|first4=Deyu|last5=Jin|first5=Qi|last6=Wang|first6=Da-Cheng|title=Crystal Structure of a Glutamate/Aspartate Binding Protein Complexed with a Glutamate Molecule: Structural Basis of Ligand Specificity at Atomic Resolution|journal=Journal of Molecular Biology|volume=382|issue=1|pages=99–111|doi=10.1016/j.jmb.2008.06.091|pmid=18640128|year=2008}}{{Cite journal|last1=De Lorimier|first1=Robert M.|last2=Smith|first2=J. Jeff|last3=Dwyer|first3=Mary A.|last4=Looger|first4=Loren L.|last5=Sali|first5=Kevin M.|last6=Paavola|first6=Chad D.|last7=Rizk|first7=Shahir S.|last8=Sadigov|first8=Shamil|last9=Conrad|first9=David W.|date=2002-11-01|title=Construction of a fluorescent biosensor family|journal=Protein Science|language=en|volume=11|issue=11|pages=2655–2675|doi=10.1110/ps.021860|pmid=12381848|pmc=2373719|issn=1469-896X}} Glutamate binding changed the distance between CFP and YFP, changing the efficiency of energy transfer (FRET) between the two fluorophores.{{Cite journal|last1=Okumoto|first1=Sakiko|last2=Looger|first2=Loren L.|last3=Micheva|first3=Kristina D.|last4=Reimer|first4=Richard J.|last5=Smith|first5=Stephen J.|last6=Frommer|first6=Wolf B.|date=2005-06-14|title=Detection of glutamate release from neurons by genetically encoded surface-displayed FRET nanosensors|journal=Proceedings of the National Academy of Sciences of the United States of America|language=en|volume=102|issue=24|pages=8740–8745|doi=10.1073/pnas.0503274102|issn=0027-8424|pmid=15939876|pmc=1143584|bibcode=2005PNAS..102.8740O|doi-access=free}}{{Cite journal|last1=Hires|first1=Samuel Andrew|last2=Zhu|first2=Yongling|last3=Tsien|first3=Roger Y.|date=2008-03-18|title=Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters|journal=Proceedings of the National Academy of Sciences|language=en|volume=105|issue=11|pages=4411–4416|doi=10.1073/pnas.0712008105|issn=0027-8424|pmid=18332427|pmc=2393813|bibcode=2008PNAS..105.4411H|doi-access=free}} A breakthrough in visualizing glutamate release was achieved with iGluSnFR, a single-fluorophore glutamate sensor based on EGFP producing a ~5‑fold increase in fluorescence. To measure synaptic transmission at high frequencies, novel iGluSnFR variants with accelerated kinetics have recently been developed.{{Cite journal|last1=Helassa|first1=Nordine|last2=Dürst|first2=Céline D.|last3=Coates|first3=Catherine|last4=Kerruth|first4=Silke|last5=Arif|first5=Urwa|last6=Schulze|first6=Christian|last7=Wiegert|first7=J. Simon|last8=Geeves|first8=Michael|last9=Oertner|first9=Thomas G.|last10=Török|first10=Katalin|date=2018-05-22|title=Ultrafast glutamate sensors resolve high-frequency release at Schaffer collateral synapses|journal=Proceedings of the National Academy of Sciences|volume=115|issue=21|pages=5594–5599|doi=10.1073/pnas.1720648115|pmc=6003469|pmid=29735711|doi-access=free}}{{Cite journal|last1=Marvin|first1=Jonathan S.|last2=Scholl|first2=Benjamin|last3=Wilson|first3=Daniel E.|last4=Podgorski|first4=Kaspar|last5=Kazemipour|first5=Abbas|last6=Müller|first6=Johannes Alexander|last7=Schoch|first7=Susanne|last8=Quiroz|first8=Francisco José Urra|last9=Rebola|first9=Nelson|last10=Bao|first10=Huan|last11=Little|first11=Justin P.|date=November 2018|title=Stability, affinity, and chromatic variants of the glutamate sensor iGluSnFR|url= |journal=Nature Methods|language=en|volume=15|issue=11|pages=936–939|doi=10.1038/s41592-018-0171-3|issn=1548-7105|pmc=6394230|pmid=30377363}}

References

Category:Neuroscience

Category:Membrane proteins

Category:Fluorescent proteins

Category:Genetic engineering