PT  - JOURNAL ARTICLE
AU  - Thomas Schlichthaerle
AU  - Maximilian T. Strauss
AU  - Florian Schueder
AU  - Alexander Auer
AU  - Bianca Nijmeijer
AU  - Moritz Kueblbeck
AU  - Vilma Jimenez Sabinina
AU  - Jervis V. Thevathasan
AU  - Jonas Ries
AU  - Jan Ellenberg
AU  - Ralf Jungmann
TI  - Direct visualization of single nuclear pore complex proteins using genetically-encoded probes for DNA-PAINT
AID  - 10.1101/579961
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 579961
4099  - http://biorxiv.org/content/early/2019/03/23/579961.short
4100  - http://biorxiv.org/content/early/2019/03/23/579961.full
AB  - 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.