Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies

Autores
Saavedra, Lucas; Buena Maizón, Héctor; Barrantes, Francisco Jose
Año de publicación
2022
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
The cell-surface topography and density of nicotinic acetylcholine receptors (nAChRs) play a key functional role in the synapse. Here we employ in parallel two labeling and two super-resolution microscopy strategies to characterize the distribution of this receptor at the plasma membrane of the mammalian clonal cell line CHO-K1/A5. Cells were interrogated with two targeted techniques (confocal microscopy and stimulated emission depletion (STED) nanoscopy) and single-molecule nanoscopy (stochastic optical reconstruction microscopy, STORM) using the same fluorophore, Alexa Fluor 647, tagged onto either α-bungarotoxin (BTX) or the monoclonal antibody mAb35. Analysis of the topography of nanometer-sized aggregates (“nanoclusters”) was carried out using STORMGraph, a quantitative clustering analysis for single-molecule localization microscopy based on graph theory and community detection, and ASTRICS, an inter-cluster similarity algorithm based on computational geometry. Antibody-induced crosslinking of receptors resulted in nanoclusters with a larger number of receptor molecules and higher densities than those observed in BTX-labeled samples. STORM and STED provided complementary information, STED rendering a direct map of the mesoscale nAChR distribution at distances ~10-times larger than the nanocluster centroid distances measured in STORM samples. By applying photon threshold filtering analysis, we show that it is also possible to detect the mesoscale organization in STORM images.
Fil: Saavedra, Lucas. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina
Fil: Buena Maizón, Héctor. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina
Fil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina
Materia
CHOLESTEROL
NANOCLUSTERS
NANOSCOPY
NICOTINIC ACETYLCHOLINE RECEPTOR
STED
STORM
SUPER-RESOLUTION MICROSCOPY
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/214676
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/214676
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM NanoscopiesSaavedra, LucasBuena Maizón, HéctorBarrantes, Francisco JoseCHOLESTEROLNANOCLUSTERSNANOSCOPYNICOTINIC ACETYLCHOLINE RECEPTORSTEDSTORMSUPER-RESOLUTION MICROSCOPYhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The cell-surface topography and density of nicotinic acetylcholine receptors (nAChRs) play a key functional role in the synapse. Here we employ in parallel two labeling and two super-resolution microscopy strategies to characterize the distribution of this receptor at the plasma membrane of the mammalian clonal cell line CHO-K1/A5. Cells were interrogated with two targeted techniques (confocal microscopy and stimulated emission depletion (STED) nanoscopy) and single-molecule nanoscopy (stochastic optical reconstruction microscopy, STORM) using the same fluorophore, Alexa Fluor 647, tagged onto either α-bungarotoxin (BTX) or the monoclonal antibody mAb35. Analysis of the topography of nanometer-sized aggregates (“nanoclusters”) was carried out using STORMGraph, a quantitative clustering analysis for single-molecule localization microscopy based on graph theory and community detection, and ASTRICS, an inter-cluster similarity algorithm based on computational geometry. Antibody-induced crosslinking of receptors resulted in nanoclusters with a larger number of receptor molecules and higher densities than those observed in BTX-labeled samples. STORM and STED provided complementary information, STED rendering a direct map of the mesoscale nAChR distribution at distances ~10-times larger than the nanocluster centroid distances measured in STORM samples. By applying photon threshold filtering analysis, we show that it is also possible to detect the mesoscale organization in STORM images.Fil: Saavedra, Lucas. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Buena Maizón, Héctor. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; ArgentinaFil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaMolecular Diversity Preservation International2022-09info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/214676Saavedra, Lucas; Buena Maizón, Héctor; Barrantes, Francisco Jose; Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies; Molecular Diversity Preservation International; International Journal of Molecular Sciences; 23; 18; 9-2022; 1-221422-0067CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.mdpi.com/1422-0067/23/18/10435info:eu-repo/semantics/altIdentifier/doi/10.3390/ijms231810435info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-10-15T14:24:06Zoai:ri.conicet.gov.ar:11336/214676instacron: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:34982025-10-15 14:24:06.832CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies
title Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies
spellingShingle Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies
Saavedra, Lucas
CHOLESTEROL
NANOCLUSTERS
NANOSCOPY
NICOTINIC ACETYLCHOLINE RECEPTOR
STED
STORM
SUPER-RESOLUTION MICROSCOPY
title_short Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies
title_full Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies
title_fullStr Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies
title_full_unstemmed Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies
title_sort Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies
dc.creator.none.fl_str_mv Saavedra, Lucas
Buena Maizón, Héctor
Barrantes, Francisco Jose
author Saavedra, Lucas
author_facet Saavedra, Lucas
Buena Maizón, Héctor
Barrantes, Francisco Jose
author_role author
author2 Buena Maizón, Héctor
Barrantes, Francisco Jose
author2_role author
author
dc.subject.none.fl_str_mv CHOLESTEROL
NANOCLUSTERS
NANOSCOPY
NICOTINIC ACETYLCHOLINE RECEPTOR
STED
STORM
SUPER-RESOLUTION MICROSCOPY
topic CHOLESTEROL
NANOCLUSTERS
NANOSCOPY
NICOTINIC ACETYLCHOLINE RECEPTOR
STED
STORM
SUPER-RESOLUTION MICROSCOPY
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv The cell-surface topography and density of nicotinic acetylcholine receptors (nAChRs) play a key functional role in the synapse. Here we employ in parallel two labeling and two super-resolution microscopy strategies to characterize the distribution of this receptor at the plasma membrane of the mammalian clonal cell line CHO-K1/A5. Cells were interrogated with two targeted techniques (confocal microscopy and stimulated emission depletion (STED) nanoscopy) and single-molecule nanoscopy (stochastic optical reconstruction microscopy, STORM) using the same fluorophore, Alexa Fluor 647, tagged onto either α-bungarotoxin (BTX) or the monoclonal antibody mAb35. Analysis of the topography of nanometer-sized aggregates (“nanoclusters”) was carried out using STORMGraph, a quantitative clustering analysis for single-molecule localization microscopy based on graph theory and community detection, and ASTRICS, an inter-cluster similarity algorithm based on computational geometry. Antibody-induced crosslinking of receptors resulted in nanoclusters with a larger number of receptor molecules and higher densities than those observed in BTX-labeled samples. STORM and STED provided complementary information, STED rendering a direct map of the mesoscale nAChR distribution at distances ~10-times larger than the nanocluster centroid distances measured in STORM samples. By applying photon threshold filtering analysis, we show that it is also possible to detect the mesoscale organization in STORM images.
Fil: Saavedra, Lucas. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina
Fil: Buena Maizón, Héctor. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina
Fil: Barrantes, Francisco Jose. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina
description The cell-surface topography and density of nicotinic acetylcholine receptors (nAChRs) play a key functional role in the synapse. Here we employ in parallel two labeling and two super-resolution microscopy strategies to characterize the distribution of this receptor at the plasma membrane of the mammalian clonal cell line CHO-K1/A5. Cells were interrogated with two targeted techniques (confocal microscopy and stimulated emission depletion (STED) nanoscopy) and single-molecule nanoscopy (stochastic optical reconstruction microscopy, STORM) using the same fluorophore, Alexa Fluor 647, tagged onto either α-bungarotoxin (BTX) or the monoclonal antibody mAb35. Analysis of the topography of nanometer-sized aggregates (“nanoclusters”) was carried out using STORMGraph, a quantitative clustering analysis for single-molecule localization microscopy based on graph theory and community detection, and ASTRICS, an inter-cluster similarity algorithm based on computational geometry. Antibody-induced crosslinking of receptors resulted in nanoclusters with a larger number of receptor molecules and higher densities than those observed in BTX-labeled samples. STORM and STED provided complementary information, STED rendering a direct map of the mesoscale nAChR distribution at distances ~10-times larger than the nanocluster centroid distances measured in STORM samples. By applying photon threshold filtering analysis, we show that it is also possible to detect the mesoscale organization in STORM images.
publishDate 2022
dc.date.none.fl_str_mv 2022-09
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/11336/214676
Saavedra, Lucas; Buena Maizón, Héctor; Barrantes, Francisco Jose; Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies; Molecular Diversity Preservation International; International Journal of Molecular Sciences; 23; 18; 9-2022; 1-22
1422-0067
CONICET Digital
CONICET
url http://hdl.handle.net/11336/214676
identifier_str_mv Saavedra, Lucas; Buena Maizón, Héctor; Barrantes, Francisco Jose; Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies; Molecular Diversity Preservation International; International Journal of Molecular Sciences; 23; 18; 9-2022; 1-22
1422-0067
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/url/https://www.mdpi.com/1422-0067/23/18/10435
info:eu-repo/semantics/altIdentifier/doi/10.3390/ijms231810435
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Molecular Diversity Preservation International
publisher.none.fl_str_mv Molecular Diversity Preservation International
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
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score 13.22299

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