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 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

Correction: Circumvention of common labelling artefacts using secondary nanobodies

Correction for ‘Circumvention of common labelling artefacts using secondary nanobodies’ by Shama Sograte-Idrissi et al., Nanoscale, 2020, 12, 10226–10239, DOI: 10.1039/D0NR00227E

The nucleolus functions as a phase-separated protein quality control compartment

The nuclear proteome is rich in stress-sensitive proteins, which suggests that effective protein quality control mechanisms are in place to ensure conformational maintenance. We investigated the role of the nucleolus in this process. In mammalian tissue culture cells under stress conditions, misfolded proteins entered the granular component (GC) phase of the nucleolus. Transient associations with nucleolar proteins such as NPM1 conferred low mobility to misfolded proteins within the liquid-like GC phase, avoiding irreversible aggregation. Refolding and extraction of proteins from the nucleolus during recovery from stress was Hsp70-dependent. The capacity of the nucleolus to store misfolded proteins was limited, and prolonged stress led to a transition of the nucleolar matrix from liquid-like to solid, with loss of reversibility and dysfunction in quality control. Thus, we suggest that the nucleolus has chaperone-like properties and can promote nuclear protein maintenance under stress.We acknowledge support by the MPIB Imaging facility and G. Cardone for providing the algorithm for image quantification

Exploiting nanobodies and Affimers for superresolution imaging in light microscopy

Antibodies have long been the main approach used for localizing proteins of interest by light microscopy. In the past 5 yr or so, and with the advent of superresolution microscopy, the diversity of tools for imaging has rapidly expanded. One main area of expansion has been in the area of nanobodies, small single-chain antibodies from camelids or sharks. The other has been the use of artificial scaffold proteins, including Affimers. The small size of nanobodies and Affimers compared with the traditional antibody provides several advantages for superresolution imaging

Super-resolved visualization of single DNA-based tension sensors in cell adhesion

Cell-extracellular matrix sensing plays a crucial role in cellular behavior and leads to the formation of a macromolecular protein complex called the focal adhesion. Despite their importance in cellular decision making, relatively little is known about cell-matrix interactions and the intracellular transduction of an initial ligand-receptor binding event on the single-molecule level. Here, we combine cRGD-ligand-decorated DNA tension sensors with DNA-PAINT super-resolution microscopy to study the mechanical engagement of single integrin receptors and the downstream influence on actin bundling. We uncover that integrin receptor clustering is governed by a non-random organization with complexes spaced at 20-30nm distances. The DNA-based tension sensor and analysis framework provide powerful tools to study a multitude of receptor-ligand interactions where forces are involved in ligand-receptor binding. Relatively little is known about cell-matrix interactions and the intracellular transduction of an initial ligand-receptor binding event on the single-molecule level. Here authors combine ligand-decorated DNA tension sensors with DNA-PAINT super-resolution microscopy to study the mechanical engagement of single integrin receptors and the downstream influence on actin bundling

Communication

Current optical super-resolution implementations are capable of resolving features spaced just a few nanometers apart. However, translating this spatial resolution to cellular targets is limited by the large size of traditionally employed primary and secondary antibody reagents. Recent advancements in small and efficient protein binders for super-resolution microscopy such as nanobodies or aptamers provide an exciting avenue for the future, however their widespread availability is still limited. To address this issue, we here report the combination of bacterial-derived binders commonly used in antibody purification with DNA-PAINT microscopy. The small size of these protein binders compared to secondary antibodies make them an attractive labeling alternative for emerging super-resolution techniques. We here present a labeling protocol for DNA conjugation of bacterial-derived protein A and G for DNA-PAINT imaging and assay their performance intracellularly by targeting primary antibodies against Tubulin, TOM20, and EGFR and quantify the increase in obtainable resolution. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.We also acknowledge the support of the imaging facility at the MPI of Biochemistry

DNA nanotechnology and fluorescence applications

Structural DNA nanotechnology allow researchers to use the unique molecular recognition properties of DNA strands to construct nanoscale objects with almost arbitrary complexity in two and three dimensions. Abstracted as molecular breadboards, DNA nanostructures enable nanometer-precise placement of guest molecules such as proteins, fluorophores, or nanoparticles. These assemblies can be used to study biological phenomena with unprecedented control over number, spacing, and molecular identity. Here, we give a general introduction to structural DNA nanotechnology and more specifically discuss applications of DNA nanostructures in the field of fluorescence and plasmonics

Quantitative Assessment of Labeling Probes for Super-Resolution Microscopy Using Designer DNA Nanostructures

Improving labeling probes for state-of-the-art super-resolution microscopy is becoming of major importance. However, there is currently a lack of tools to quantitatively evaluate probe performance regarding efficiency, precision, and achievable resolution in an unbiased yet modular fashion. Herein, we introduce designer DNA origami structures combined with DNA-PAINT to overcome this issue and evaluate labeling efficiency, precision, and quantification using antibodies and nanobodies as exemplary labeling probes. Whereas current assessment of binders is mostly qualitative, e. g. based on an expected staining pattern, we herein present a quantitative analysis platform of the antigen labeling efficiency and achievable resolution, allowing researchers to choose the best performing binder. The platform can furthermore be readily adapted for discovery and precise quantification of a large variety of additional labeling probes

Super-resolution microscopy with DNA-PAINT

Super-resolution techniques have begun to transform biological and biomedical research by allowing researchers to observe structures well below the classic diffraction limit of light. DNA points accumulation for imaging in nanoscale topography (DNA PAINT) offers an easy-to-implement approach to localization-based super-resolution microscopy, owing to the use of DNA probes. In DNA-PAINT, transient binding of short dye-labeled ('imager') oligonucleotides to their complementary target ('docking') strands creates the necessary 'blinking to enable stochastic super-resolution microscopy. Using the programmability and specificity of DNA molecules as imaging and labeling probes allows researchers to decouple blinking from dye photophysics, alleviating limitations of current super-resolution techniques, making them compatible with virtually any single-molecule-compatible dye. Recent developments in DNA-PAINT have enabled spectrally unlimited multiplexing, precise molecule counting and ultra-high, molecular scale (sub-5-nm) spatial resolution, reaching 1-nm localization precision. DNA-PAINT can be applied to a multitude of in vitro and cellular applications by linking docking strands to antibodies. Here, we present a protocol for the key aspects of the DNA PAINT framework for both novice and expert users. This protocol describes the creation of DNA origami test samples, in situ sample preparation, multiplexed data acquisition, data simulation, super-resolution image reconstruction and post-processing such as drift correction, molecule counting (qPAINT) and particle averaging. Moreover, we provide an integrated software package, named Picasso, for the computational steps involved. The protocol is designed to be modular, so that individual components can be chosen and implemented per requirements of a specific application. The procedure can be completed in 1-2 d

Fast, Background-Free DNA-PAINT Imaging Using FRET-Based Probes

DNA point accumulation in nanoscale topography (DNA-PAINT) enables super-resolution microscopy by harnessing the predictable, transient hybridization between short dye-labeled “imager” and complementary target-bound “docking” strands. DNA-PAINT microscopy allows sub-5 nm spatial resolution, spectrally unlimited multiplexing, and quantitative image analysis. However, these abilities come at the cost of nonfluorogenic imager strands, also emitting fluorescence when not bound to their docking strands. This has thus far prevented rapid image acquisition with DNA-PAINT, as the blinking rate of probes is limited by an upper-bound of imager strand concentrations, which in turn is dictated by the necessity to facilitate the detection of single-molecule binding events over the background of unbound, freely diffusing probes. To overcome this limitation and enable fast, background-free DNA-PAINT microscopy, we here introduce FRET-based imaging probes, alleviating the concentration-limit of imager strands and speeding up image acquisition by several orders of magnitude. We assay two approaches for FRET-based DNA-PAINT (or FRET-PAINT) using either fixed or transient acceptor dyes in combination with transiently binding donor-labeled DNA strands and achieve high-quality super-resolution imaging on DNA origami structures in a few tens of seconds. Finally, we also demonstrate the applicability of FRET-PAINT in a cellular environment by performing super-resolution imaging of microtubules in under 30 s. FRET-PAINT combines the advantages of conventional DNA-PAINT with fast image acquisition times, facilitating the potential study of dynamic processes