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

Thomas Schlichthaerle,Vilma Jimenez Sabinina,Jervis V Thevathasan,Bianca Nijmeijer,Florian Schueder,Maximilian T Strauss,Ralf Jungmann,Moritz Kueblbeck,Jonas Ries,Alexander Auer,Jan Ellenberg

doi:10.1002/anie.201905685

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 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.

Highlights

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 nuclear pore complex (NPC) complex
While we are convinced that some of these issues might be resolved in the future, we introduce the combination of widely-used, genetically-encoded self-labeling enzymes such as SNAP-tag[4] and HaloTag[5] with DNA-PAINT to enable 1:1 labeling of single proteins in the nuclear pore complex (NPC) using ligand-conjugated DNA-PAINT docking strands
We investigate the achievable labeling precision and reduction of linkage error of SNAP-tag and HaloTag, examine their performance in contrast to DNA-conjugated nanobodies against GFP-tagged proteins in single NPCs and further compare them to primary and secondary antibody labeling

Summary

Introduction

We combine genetically-encoded self-labeling enzymes such as SNAP-tag and HaloTag with DNA-PAINT microscopy. While we are convinced that some of these issues might be resolved in the future, we introduce the combination of widely-used, genetically-encoded self-labeling enzymes such as SNAP-tag[4] and HaloTag[5] with DNA-PAINT to enable 1:1 labeling of single proteins in the nuclear pore complex (NPC) using ligand-conjugated DNA-PAINT docking strands.

Results

Conclusion