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Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT

Thomas Schlichthaerle, Maximilian T. Strauss, Florian Schueder, Alexander Auer, Bianca Nijmeijer, Moritz Kueblbeck, Vilma Jimenez Sabinina, Jervis V. Thevathasan, View ORCID ProfileJonas Ries, View ORCID ProfileJan Ellenberg, View ORCID ProfileRalf Jungmann
doi: https://doi.org/10.1101/579961
Thomas Schlichthaerle
1Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.
2Max Planck Institute of Biochemistry, Martinsried, Germany.
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Maximilian T. Strauss
1Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.
2Max Planck Institute of Biochemistry, Martinsried, Germany.
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Florian Schueder
1Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.
2Max Planck Institute of Biochemistry, Martinsried, Germany.
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Alexander Auer
1Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.
2Max Planck Institute of Biochemistry, Martinsried, Germany.
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Bianca Nijmeijer
3Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
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Moritz Kueblbeck
3Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
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Vilma Jimenez Sabinina
3Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
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Jervis V. Thevathasan
3Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
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Jonas Ries
3Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
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  • ORCID record for Jonas Ries
Jan Ellenberg
3Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany.
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  • ORCID record for Jan Ellenberg
Ralf Jungmann
1Faculty of Physics and Center for Nanoscience, Ludwig Maximilian University, Munich, Germany.
2Max Planck Institute of Biochemistry, Martinsried, Germany.
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  • ORCID record for Ralf Jungmann
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Abstract

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 superresolution studies advancing our understanding of the NPC's complex architecture. However, the specific and direct visualization of single copies of NPC proteins and thus the ability to observe single-molecule heterogeneities of these complex structures is thus far elusive. Here, we combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. We employ the high localization precision in DNA-PAINT and molecular contrast of these protein tags to optically resolve single copies of nucleoporins in the human Y-complex in three dimensions with a precision of ~3 nm. This technological advancement now enables structural studies of multicomponent complexes on the level of single proteins in cells using optical fluorescence microscopy.

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Posted March 23, 2019.
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Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT
Thomas Schlichthaerle, Maximilian T. Strauss, Florian Schueder, Alexander Auer, Bianca Nijmeijer, Moritz Kueblbeck, Vilma Jimenez Sabinina, Jervis V. Thevathasan, Jonas Ries, Jan Ellenberg, Ralf Jungmann
bioRxiv 579961; doi: https://doi.org/10.1101/579961
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Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT
Thomas Schlichthaerle, Maximilian T. Strauss, Florian Schueder, Alexander Auer, Bianca Nijmeijer, Moritz Kueblbeck, Vilma Jimenez Sabinina, Jervis V. Thevathasan, Jonas Ries, Jan Ellenberg, Ralf Jungmann
bioRxiv 579961; doi: https://doi.org/10.1101/579961

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