An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging
Autor(a) principal: | |
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Data de Publicação: | 2013 |
Outros Autores: | |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10316/109788 https://doi.org/10.1371/journal.pone.0052874 |
Resumo: | Ras is a signaling protein involved in a variety of cellular processes. Hence, studying Ras signaling with high spatiotemporal resolution is crucial to understanding the roles of Ras in many important cellular functions. Previously, fluorescence lifetime imaging (FLIM) of fluorescent resonance energy transfer (FRET)-based Ras activity sensors, FRas and FRas-F, have been demonstrated to be useful for measuring the spatiotemporal dynamics of Ras signaling in subcellular micro-compartments. However the predominantly nuclear localization of the sensors' acceptor has limited its sensitivity. Here, we have overcome this limitation and developed two variants of the existing FRas sensor with different affinities: FRas2-F (K(d)∼1.7 µM) and FRas2-M (K(d)∼0.5 µM). We demonstrate that, under 2-photon fluorescence lifetime imaging microscopy, FRas2 sensors provide higher sensitivity compared to previous sensors in 293T cells and neurons. |
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An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imagingAmino Acid SequenceAnimalsBiosensing TechniquesEnzyme ActivationHEK293 CellsHumansMolecular Sequence DataMutationProtein Structure, TertiaryProto-Oncogene Proteins c-rafRatsRats, Sprague-Dawleyras ProteinsFluorescence Resonance Energy TransferMicroscopy, Fluorescence, MultiphotonRas is a signaling protein involved in a variety of cellular processes. Hence, studying Ras signaling with high spatiotemporal resolution is crucial to understanding the roles of Ras in many important cellular functions. Previously, fluorescence lifetime imaging (FLIM) of fluorescent resonance energy transfer (FRET)-based Ras activity sensors, FRas and FRas-F, have been demonstrated to be useful for measuring the spatiotemporal dynamics of Ras signaling in subcellular micro-compartments. However the predominantly nuclear localization of the sensors' acceptor has limited its sensitivity. Here, we have overcome this limitation and developed two variants of the existing FRas sensor with different affinities: FRas2-F (K(d)∼1.7 µM) and FRas2-M (K(d)∼0.5 µM). We demonstrate that, under 2-photon fluorescence lifetime imaging microscopy, FRas2 sensors provide higher sensitivity compared to previous sensors in 293T cells and neurons.Public Library of Science2013info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://hdl.handle.net/10316/109788http://hdl.handle.net/10316/109788https://doi.org/10.1371/journal.pone.0052874eng1932-6203Oliveira, Ana F.Yasuda, Ryoheiinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-10-26T10:51:24Zoai:estudogeral.uc.pt:10316/109788Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T21:25:56.162834Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging |
title |
An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging |
spellingShingle |
An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging Oliveira, Ana F. Amino Acid Sequence Animals Biosensing Techniques Enzyme Activation HEK293 Cells Humans Molecular Sequence Data Mutation Protein Structure, Tertiary Proto-Oncogene Proteins c-raf Rats Rats, Sprague-Dawley ras Proteins Fluorescence Resonance Energy Transfer Microscopy, Fluorescence, Multiphoton |
title_short |
An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging |
title_full |
An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging |
title_fullStr |
An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging |
title_full_unstemmed |
An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging |
title_sort |
An improved Ras sensor for highly sensitive and quantitative FRET-FLIM imaging |
author |
Oliveira, Ana F. |
author_facet |
Oliveira, Ana F. Yasuda, Ryohei |
author_role |
author |
author2 |
Yasuda, Ryohei |
author2_role |
author |
dc.contributor.author.fl_str_mv |
Oliveira, Ana F. Yasuda, Ryohei |
dc.subject.por.fl_str_mv |
Amino Acid Sequence Animals Biosensing Techniques Enzyme Activation HEK293 Cells Humans Molecular Sequence Data Mutation Protein Structure, Tertiary Proto-Oncogene Proteins c-raf Rats Rats, Sprague-Dawley ras Proteins Fluorescence Resonance Energy Transfer Microscopy, Fluorescence, Multiphoton |
topic |
Amino Acid Sequence Animals Biosensing Techniques Enzyme Activation HEK293 Cells Humans Molecular Sequence Data Mutation Protein Structure, Tertiary Proto-Oncogene Proteins c-raf Rats Rats, Sprague-Dawley ras Proteins Fluorescence Resonance Energy Transfer Microscopy, Fluorescence, Multiphoton |
description |
Ras is a signaling protein involved in a variety of cellular processes. Hence, studying Ras signaling with high spatiotemporal resolution is crucial to understanding the roles of Ras in many important cellular functions. Previously, fluorescence lifetime imaging (FLIM) of fluorescent resonance energy transfer (FRET)-based Ras activity sensors, FRas and FRas-F, have been demonstrated to be useful for measuring the spatiotemporal dynamics of Ras signaling in subcellular micro-compartments. However the predominantly nuclear localization of the sensors' acceptor has limited its sensitivity. Here, we have overcome this limitation and developed two variants of the existing FRas sensor with different affinities: FRas2-F (K(d)∼1.7 µM) and FRas2-M (K(d)∼0.5 µM). We demonstrate that, under 2-photon fluorescence lifetime imaging microscopy, FRas2 sensors provide higher sensitivity compared to previous sensors in 293T cells and neurons. |
publishDate |
2013 |
dc.date.none.fl_str_mv |
2013 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10316/109788 http://hdl.handle.net/10316/109788 https://doi.org/10.1371/journal.pone.0052874 |
url |
http://hdl.handle.net/10316/109788 https://doi.org/10.1371/journal.pone.0052874 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
1932-6203 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.publisher.none.fl_str_mv |
Public Library of Science |
publisher.none.fl_str_mv |
Public Library of Science |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
instname_str |
Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
instacron_str |
RCAAP |
institution |
RCAAP |
reponame_str |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
collection |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
repository.name.fl_str_mv |
Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
repository.mail.fl_str_mv |
|
_version_ |
1799134140888514560 |