Direct Visualization of Single Nuclear Pore Complex Proteins Using Genetically‐Encoded Probes for DNA‐PAINT

The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cr...

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Bibliographic Details
Published in:Angewandte Chemie 2019-09, Vol.131 (37), p.13138-13142
Main Authors: Schlichthaerle, Thomas, Strauss, Maximilian T., Schueder, Florian, Auer, Alexander, Nijmeijer, Bianca, Kueblbeck, Moritz, Jimenez Sabinina, Vilma, Thevathasan, Jervis V., Ries, Jonas, Ellenberg, Jan, Jungmann, Ralf
Format: Article
Language:English
Subjects:
Architecture
Chemistry
Deoxyribonucleic acid
DNA
DNA probes
DNA-PAINT
Einzelmolekül-Bildgebung
Electron microscopy
Fluorescence
Fluorescence microscopy
Genetisch kodierte Marker
Kernporenkomplex
Microscopy
Nucleoporins
Proteins
Superauflösende Mikroskopie
Visualization
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Summary:The nuclear pore complex (NPC) is one of the largest and most complex protein assemblies in the cell and, among other functions, serves as the gatekeeper of nucleocytoplasmic transport. Unraveling its molecular architecture and functioning has been an active research topic for decades with recent cryogenic electron microscopy and super‐resolution studies advancing our understanding of the architecture of the NPC complex. However, the specific and direct visualization of single copies of NPC proteins is thus far elusive. Herein, we combine genetically‐encoded self‐labeling enzymes such as SNAP‐tag and HaloTag with DNA‐PAINT microscopy. We resolve single copies of nucleoporins in the human Y‐complex in three dimensions with a precision of circa 3 nm, enabling studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy. Tiefer Einblick in die Zelle: Genetisch kodierte selbstmarkierende Enzyme wie z. B. SNAP‐tag und HaloTag werden mit DNA‐PAINT‐Mikroskopie kombiniert. Dies ermöglicht die Untersuchung von Multikomponentenkomplexen in Zellen auf dem Niveau einzelner Proteine.
ISSN:0044-8249
1521-3757
DOI:10.1002/ange.201905685