Quantifying transcription factor binding dynamics at the single-molecule level in live cells

Autores
Presman, Diego Martin; Ball, David A.; Paakinaho, Ville; Grimm, Jonathan B.; Lavis, Luke D.; Karpova, Tatiana S.; Hager, Gordon L.
Año de publicación
2017
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.
Fil: Presman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. National Institutes of Health; Estados Unidos
Fil: Ball, David A.. National Institutes of Health; Estados Unidos
Fil: Paakinaho, Ville. National Institutes of Health; Estados Unidos
Fil: Grimm, Jonathan B.. Howard Hughes Medical Institute; Estados Unidos
Fil: Lavis, Luke D.. Howard Hughes Medical Institute; Estados Unidos
Fil: Karpova, Tatiana S.. National Institutes of Health; Estados Unidos
Fil: Hager, Gordon L.. National Institutes of Health; Estados Unidos
Materia
DNA BINDING
DYNAMICS
FLUORESCENCE MICROSCOPY
GLUCOCORTICOID RECEPTOR
SINGLE-MOLECULE TRACKING
TRANSCRIPTION FACTOR
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/64420
Acceder
id CONICETDig_388812089c44ed85216bdc69e2510070
oai_identifier_str oai:ri.conicet.gov.ar:11336/64420
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Quantifying transcription factor binding dynamics at the single-molecule level in live cellsPresman, Diego MartinBall, David A.Paakinaho, VilleGrimm, Jonathan B.Lavis, Luke D.Karpova, Tatiana S.Hager, Gordon L.DNA BINDINGDYNAMICSFLUORESCENCE MICROSCOPYGLUCOCORTICOID RECEPTORSINGLE-MOLECULE TRACKINGTRANSCRIPTION FACTORhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.Fil: Presman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. National Institutes of Health; Estados UnidosFil: Ball, David A.. National Institutes of Health; Estados UnidosFil: Paakinaho, Ville. National Institutes of Health; Estados UnidosFil: Grimm, Jonathan B.. Howard Hughes Medical Institute; Estados UnidosFil: Lavis, Luke D.. Howard Hughes Medical Institute; Estados UnidosFil: Karpova, Tatiana S.. National Institutes of Health; Estados UnidosFil: Hager, Gordon L.. National Institutes of Health; Estados UnidosAcademic Press Inc Elsevier Science2017-07info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/64420Presman, Diego Martin; Ball, David A.; Paakinaho, Ville; Grimm, Jonathan B.; Lavis, Luke D.; et al.; Quantifying transcription factor binding dynamics at the single-molecule level in live cells; Academic Press Inc Elsevier Science; Methods; 123; 7-2017; 76-881046-2023CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.ymeth.2017.03.014info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1046202316304509info:eu-repo/semantics/altIdentifier/url/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522764/info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T09:53:19Zoai:ri.conicet.gov.ar:11336/64420instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 09:53:19.352CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Quantifying transcription factor binding dynamics at the single-molecule level in live cells
title Quantifying transcription factor binding dynamics at the single-molecule level in live cells
spellingShingle Quantifying transcription factor binding dynamics at the single-molecule level in live cells
Presman, Diego Martin
DNA BINDING
DYNAMICS
FLUORESCENCE MICROSCOPY
GLUCOCORTICOID RECEPTOR
SINGLE-MOLECULE TRACKING
TRANSCRIPTION FACTOR
title_short Quantifying transcription factor binding dynamics at the single-molecule level in live cells
title_full Quantifying transcription factor binding dynamics at the single-molecule level in live cells
title_fullStr Quantifying transcription factor binding dynamics at the single-molecule level in live cells
title_full_unstemmed Quantifying transcription factor binding dynamics at the single-molecule level in live cells
title_sort Quantifying transcription factor binding dynamics at the single-molecule level in live cells
dc.creator.none.fl_str_mv Presman, Diego Martin
Ball, David A.
Paakinaho, Ville
Grimm, Jonathan B.
Lavis, Luke D.
Karpova, Tatiana S.
Hager, Gordon L.
author Presman, Diego Martin
author_facet Presman, Diego Martin
Ball, David A.
Paakinaho, Ville
Grimm, Jonathan B.
Lavis, Luke D.
Karpova, Tatiana S.
Hager, Gordon L.
author_role author
author2 Ball, David A.
Paakinaho, Ville
Grimm, Jonathan B.
Lavis, Luke D.
Karpova, Tatiana S.
Hager, Gordon L.
author2_role author
author
author
author
author
author
dc.subject.none.fl_str_mv DNA BINDING
DYNAMICS
FLUORESCENCE MICROSCOPY
GLUCOCORTICOID RECEPTOR
SINGLE-MOLECULE TRACKING
TRANSCRIPTION FACTOR
topic DNA BINDING
DYNAMICS
FLUORESCENCE MICROSCOPY
GLUCOCORTICOID RECEPTOR
SINGLE-MOLECULE TRACKING
TRANSCRIPTION FACTOR
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.
Fil: Presman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina. National Institutes of Health; Estados Unidos
Fil: Ball, David A.. National Institutes of Health; Estados Unidos
Fil: Paakinaho, Ville. National Institutes of Health; Estados Unidos
Fil: Grimm, Jonathan B.. Howard Hughes Medical Institute; Estados Unidos
Fil: Lavis, Luke D.. Howard Hughes Medical Institute; Estados Unidos
Fil: Karpova, Tatiana S.. National Institutes of Health; Estados Unidos
Fil: Hager, Gordon L.. National Institutes of Health; Estados Unidos
description Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.
publishDate 2017
dc.date.none.fl_str_mv 2017-07
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/64420
Presman, Diego Martin; Ball, David A.; Paakinaho, Ville; Grimm, Jonathan B.; Lavis, Luke D.; et al.; Quantifying transcription factor binding dynamics at the single-molecule level in live cells; Academic Press Inc Elsevier Science; Methods; 123; 7-2017; 76-88
1046-2023
CONICET Digital
CONICET
url http://hdl.handle.net/11336/64420
identifier_str_mv Presman, Diego Martin; Ball, David A.; Paakinaho, Ville; Grimm, Jonathan B.; Lavis, Luke D.; et al.; Quantifying transcription factor binding dynamics at the single-molecule level in live cells; Academic Press Inc Elsevier Science; Methods; 123; 7-2017; 76-88
1046-2023
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.ymeth.2017.03.014
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S1046202316304509
info:eu-repo/semantics/altIdentifier/url/https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5522764/
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
application/pdf
dc.publisher.none.fl_str_mv Academic Press Inc Elsevier Science
publisher.none.fl_str_mv Academic Press Inc Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1842269215590973440
score 13.13397

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