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

By 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

Posted 19 Mar 2019
bioRxiv DOI: 10.1101/579961 (published DOI: 10.1002/anie.201905685)

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 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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