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Red Photoactivatable Genetic Optical-Indicators
Emerging genetically-encoded Ca(2+)-indicators (GECIs) are intensiometric reporters that increase in fluorescence when bound to Ca(2+); highly suited for studying calcium-signaling in many cell types, notably neurons. Today, major efforts are devoted toward optimizing red-emitting [red fluorescent p...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270359/ https://www.ncbi.nlm.nih.gov/pubmed/32547366 http://dx.doi.org/10.3389/fncel.2020.00113 |
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author | Hussein, Wessal Berlin, Shai |
author_facet | Hussein, Wessal Berlin, Shai |
author_sort | Hussein, Wessal |
collection | PubMed |
description | Emerging genetically-encoded Ca(2+)-indicators (GECIs) are intensiometric reporters that increase in fluorescence when bound to Ca(2+); highly suited for studying calcium-signaling in many cell types, notably neurons. Today, major efforts are devoted toward optimizing red-emitting [red fluorescent protein (RFP)-based] GECIs (R-GECI), as these provide several advantages over GFP-based reporters, for instance, increased imaging depth, reduced photodamage by longer imaging wavelengths and, in principle, are better suited for use with prevalent blue-absorbing optogenetic tools (e.g., channelrhodopsin). However, excessive fluorescence from intersecting neighboring cells in very dense tissues, notably the brain, hinders the ability to collect signals from single cells and their processes. This challenge can be addressed by photoactivatable (PA) fluorescent proteins that can be rendered fluorescent on demand by user-defined targeted light. This allows activation and, thereby, collection of fluorescent signals exclusively from desired cells and their processes, while leaving all neighboring cells in the dark (i.e., non-fluorescent). Nevertheless, there are no PA R-GECIs. Here, we sought to develop PA-R-GECIs. To do so, we initially explored a recently discovered phenomenon of Ca(2+)-independent increases in fluorescence (i.e., artifacts) in an emerging R-GECI, which has led us to rationally engineer several functional PA-R-GECIs. We also take advantage of our findings to quickly engineer a novel PA-RFP, namely, PA-mRuby3. |
format | Online Article Text |
id | pubmed-7270359 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-72703592020-06-15 Red Photoactivatable Genetic Optical-Indicators Hussein, Wessal Berlin, Shai Front Cell Neurosci Neuroscience Emerging genetically-encoded Ca(2+)-indicators (GECIs) are intensiometric reporters that increase in fluorescence when bound to Ca(2+); highly suited for studying calcium-signaling in many cell types, notably neurons. Today, major efforts are devoted toward optimizing red-emitting [red fluorescent protein (RFP)-based] GECIs (R-GECI), as these provide several advantages over GFP-based reporters, for instance, increased imaging depth, reduced photodamage by longer imaging wavelengths and, in principle, are better suited for use with prevalent blue-absorbing optogenetic tools (e.g., channelrhodopsin). However, excessive fluorescence from intersecting neighboring cells in very dense tissues, notably the brain, hinders the ability to collect signals from single cells and their processes. This challenge can be addressed by photoactivatable (PA) fluorescent proteins that can be rendered fluorescent on demand by user-defined targeted light. This allows activation and, thereby, collection of fluorescent signals exclusively from desired cells and their processes, while leaving all neighboring cells in the dark (i.e., non-fluorescent). Nevertheless, there are no PA R-GECIs. Here, we sought to develop PA-R-GECIs. To do so, we initially explored a recently discovered phenomenon of Ca(2+)-independent increases in fluorescence (i.e., artifacts) in an emerging R-GECI, which has led us to rationally engineer several functional PA-R-GECIs. We also take advantage of our findings to quickly engineer a novel PA-RFP, namely, PA-mRuby3. Frontiers Media S.A. 2020-05-28 /pmc/articles/PMC7270359/ /pubmed/32547366 http://dx.doi.org/10.3389/fncel.2020.00113 Text en Copyright © 2020 Hussein and Berlin. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Neuroscience Hussein, Wessal Berlin, Shai Red Photoactivatable Genetic Optical-Indicators |
title | Red Photoactivatable Genetic Optical-Indicators |
title_full | Red Photoactivatable Genetic Optical-Indicators |
title_fullStr | Red Photoactivatable Genetic Optical-Indicators |
title_full_unstemmed | Red Photoactivatable Genetic Optical-Indicators |
title_short | Red Photoactivatable Genetic Optical-Indicators |
title_sort | red photoactivatable genetic optical-indicators |
topic | Neuroscience |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7270359/ https://www.ncbi.nlm.nih.gov/pubmed/32547366 http://dx.doi.org/10.3389/fncel.2020.00113 |
work_keys_str_mv | AT husseinwessal redphotoactivatablegeneticopticalindicators AT berlinshai redphotoactivatablegeneticopticalindicators |