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Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells

Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intrac...

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Autores principales: Schneider, Anselm F. L., Benz, Laila S., Lehmann, Martin, Hackenberger, Christian P. R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8518916/
https://www.ncbi.nlm.nih.gov/pubmed/34288299
http://dx.doi.org/10.1002/anie.202103068
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author Schneider, Anselm F. L.
Benz, Laila S.
Lehmann, Martin
Hackenberger, Christian P. R.
author_facet Schneider, Anselm F. L.
Benz, Laila S.
Lehmann, Martin
Hackenberger, Christian P. R.
author_sort Schneider, Anselm F. L.
collection PubMed
description Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi‐synthetic nanobodies are equipped with a cleavable cell‐penetrating peptide for efficient cellular entry, which enables super‐resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis.
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spelling pubmed-85189162021-10-21 Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells Schneider, Anselm F. L. Benz, Laila S. Lehmann, Martin Hackenberger, Christian P. R. Angew Chem Int Ed Engl Research Articles Super‐resolution microscopy in living cells can be restricted by the availability of small molecule probes, which only exist against few targets and genetically encoded tags. Here, we expand the applicability of live‐cell STED by engineering cell‐permeable and highly fluorescent nanobodies as intracellular targeting agents. To ensure bright fluorescent signals at low concentrations we used the concept of intramolecular photostabilization by ligating a fluorophore along with the photostabilizer trolox to the nanobody using expressed protein ligation (EPL). Furthermore, these semi‐synthetic nanobodies are equipped with a cleavable cell‐penetrating peptide for efficient cellular entry, which enables super‐resolution imaging of GFP and mCherry, as well as two endogenous targets, nuclear lamins and the DNA replication and repair protein PCNA. We monitored cell division and DNA replication via confocal and STED microscopy thus demonstrating the utility of these new intracellular tools for functional analysis. John Wiley and Sons Inc. 2021-08-27 2021-09-27 /pmc/articles/PMC8518916/ /pubmed/34288299 http://dx.doi.org/10.1002/anie.202103068 Text en © 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH https://creativecommons.org/licenses/by-nc/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes.
spellingShingle Research Articles
Schneider, Anselm F. L.
Benz, Laila S.
Lehmann, Martin
Hackenberger, Christian P. R.
Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells
title Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells
title_full Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells
title_fullStr Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells
title_full_unstemmed Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells
title_short Cell‐Permeable Nanobodies Allow Dual‐Color Super‐Resolution Microscopy in Untransfected Living Cells
title_sort cell‐permeable nanobodies allow dual‐color super‐resolution microscopy in untransfected living cells
topic Research Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8518916/
https://www.ncbi.nlm.nih.gov/pubmed/34288299
http://dx.doi.org/10.1002/anie.202103068
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