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Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes

Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare a...

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Autores principales: Engel, Leeya, Zaoralova, Magda, Dunn, Alexander R., Oliver, Stefan
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory 2023
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10418515/
https://www.ncbi.nlm.nih.gov/pubmed/37577490
http://dx.doi.org/10.1101/2023.08.05.552110
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author Engel, Leeya
Zaoralova, Magda
Dunn, Alexander R.
Oliver, Stefan
author_facet Engel, Leeya
Zaoralova, Magda
Dunn, Alexander R.
Oliver, Stefan
author_sort Engel, Leeya
collection PubMed
description Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well-positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high resolution imaging of other adherent and non-adherent cell types with cryo-ET.
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spelling pubmed-104185152023-08-12 Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes Engel, Leeya Zaoralova, Magda Dunn, Alexander R. Oliver, Stefan bioRxiv Article Cryogenic-electron tomography (cryo-ET) has provided an unprecedented glimpse into the nanoscale architecture of cells by combining cryogenic preservation of biological structures with electron tomography. Micropatterning of extracellular matrix proteins is increasingly used as a method to prepare adherent cell types for cryo-ET as it promotes optimal positioning of cells and subcellular regions of interest for vitrification, cryo-focused ion beam (cryo-FIB) milling, and data acquisition. Here we demonstrate a micropatterning workflow for capturing minimally adherent cell types, human T-cells and Jurkat cells, for cryo-FIB and cryo-ET. Our affinity capture system facilitated the nanoscale imaging of Jurkat cells, revealing extracellular filamentous structures. It improved workflow efficiency by consistently producing grids with a sufficient number of well-positioned cells for an entire cryo-FIB session. Affinity capture can be extended to facilitate high resolution imaging of other adherent and non-adherent cell types with cryo-ET. Cold Spring Harbor Laboratory 2023-08-06 /pmc/articles/PMC10418515/ /pubmed/37577490 http://dx.doi.org/10.1101/2023.08.05.552110 Text en https://creativecommons.org/licenses/by-nc-nd/4.0/This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (https://creativecommons.org/licenses/by-nc-nd/4.0/) , which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
spellingShingle Article
Engel, Leeya
Zaoralova, Magda
Dunn, Alexander R.
Oliver, Stefan
Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes
title Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes
title_full Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes
title_fullStr Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes
title_full_unstemmed Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes
title_short Extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes
title_sort extracellular filaments revealed by affinity capture cryo-electron tomography of lymphocytes
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10418515/
https://www.ncbi.nlm.nih.gov/pubmed/37577490
http://dx.doi.org/10.1101/2023.08.05.552110
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