Nanobodies against the myelin enzyme CNPase as tools for structural and functional studies.

2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) is an abundant constituent of central nervous system non-compact myelin, frequently used as a marker antigen for myelinating cells. The catalytic activity of CNPase, the 3'-hydrolysis of 2',3'-cyclic nucleotides, is well characterised in vitro, but the in vivo function of CNPase remains unclear. CNPase interacts with the actin cytoskeleton to counteract the developmental closure of cytoplasmic channels that travel through compact myelin; its enzymatic activity may be involved in adenosine metabolism and RNA degradation. We developed a set of high-affinity nanobodies recognizing the phosphodiesterase domain of CNPase, and the crystal structures of each complex show that the five nanobodies have distinct epitopes. One of the nanobodies bound deep into the CNPase active site and acted as an inhibitor. Moreover, the nanobodies were characterised in imaging applications and as intrabodies, expressed in mammalian cells, such as primary oligodendrocytes. Fluorescently labelled nanobodies functioned in imaging of teased nerve fibers and whole brain tissue sections, as well as super-resolution microscopy. These anti-CNPase nanobodies provide new tools for structural and functional biology of myelination, including high-resolution imaging of nerve tissue.

Spatial proteomics in neurons at single-protein resolution.

To understand biological processes, it is necessary to reveal the molecular heterogeneity of cells by gaining access to the location and interaction of all biomolecules. Significant advances were achieved by super-resolution microscopy, but such methods are still far from reaching the multiplexing capacity of proteomics. Here, we introduce secondary label-based unlimited multiplexed DNA-PAINT (SUM-PAINT), a high-throughput imaging method that is capable of achieving virtually unlimited multiplexing at better than 15 nm resolution. Using SUM-PAINT, we generated 30-plex single-molecule resolved datasets in neurons and adapted omics-inspired analysis for data exploration. This allowed us to reveal the complexity of synaptic heterogeneity, leading to the discovery of a distinct synapse type. We not only provide a resource for researchers, but also an integrated acquisition and analysis workflow for comprehensive spatial proteomics at single-protein resolution.

A Versatile Synaptotagmin-1 Nanobody Provides Perturbation-Free Live Synaptic Imaging And Low Linkage-Error in Super-Resolution Microscopy.

Imaging of living synapses has relied for over two decades on the overexpression of synaptic proteins fused to fluorescent reporters. This strategy alters the stoichiometry of synaptic components and ultimately affects synapse physiology. To overcome these limitations, here a nanobody is presented that binds the calcium sensor synaptotagmin-1 (NbSyt1). This nanobody functions as an intrabody (iNbSyt1) in living neurons and is minimally invasive, leaving synaptic transmission almost unaffected, as suggested by the crystal structure of the NbSyt1 bound to Synaptotagmin-1 and by the physiological data. Its single-domain nature enables the generation of protein-based fluorescent reporters, as showcased here by measuring spatially localized presynaptic Ca2+ with a NbSyt1- jGCaMP8 chimera. Moreover, the small size of NbSyt1 makes it ideal for various super-resolution imaging methods. Overall, NbSyt1 is a versatile binder that will enable imaging in cellular and molecular neuroscience with unprecedented precision across multiple spatiotemporal scales.

An iodine-containing probe as a tool for molecular detection in secondary ion mass spectrometry.

We developed here an iodine-containing probe that can be used to identify the molecules of interest in secondary ion mass spectrometry (SIMS) by simple immunolabelling procedures. The immunolabelled iodine probe was readily combined with previously-developed SIMS probes carrying fluorine, to generate dual-channel SIMS data. This probe should provide a useful complement to the currently available SIMS probes, thus expanding the scope of this technology.

Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells.

DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.

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.

Circumvention of common labelling artefacts using secondary nanobodies.

A standard procedure to study cellular elements is via immunostaining followed by optical imaging. This methodology typically requires target-specific primary antibodies (1.Abs), which are revealed by secondary antibodies (2.Abs). Unfortunately, the antibody bivalency, polyclonality, and large size can result in a series of artifacts. Alternatively, small, monovalent probes, such as single-domain antibodies (nanobodies) have been suggested to minimize these limitations. The discovery and validation of nanobodies against specific targets are challenging, thus only a minimal amount of them are currently available. Here, we used STED, DNA-PAINT, and light-sheet microscopy, to demonstrate that secondary nanobodies (1) increase localization accuracy compared to 2.Abs; (2) allow direct pre-mixing with 1.Abs before staining, reducing experimental time, and enabling the use of multiple 1.Abs from the same species; (3) penetrate thick tissues more efficiently; and (4) avoid probe-induced clustering of target molecules observed with conventional 2.Abs in living or poorly fixed samples. Altogether, we show how secondary nanobodies are a valuable alternative to 2.Abs.

The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications.

Specialized epitope tags are widely used for detecting, manipulating or purifying proteins, but often their versatility is limited. Here, we introduce the ALFA-tag, a rationally designed epitope tag that serves a remarkably broad spectrum of applications in life sciences while outperforming established tags like the HA-, FLAG®- or myc-tag. The ALFA-tag forms a small and stable α-helix that is functional irrespective of its position on the target protein in prokaryotic and eukaryotic hosts. We characterize a nanobody (NbALFA) binding ALFA-tagged proteins from native or fixed specimen with low picomolar affinity. It is ideally suited for super-resolution microscopy, immunoprecipitations and Western blotting, and also allows in vivo detection of proteins. We show the crystal structure of the complex that enabled us to design a nanobody mutant (NbALFAPE) that permits efficient one-step purifications of native ALFA-tagged proteins, complexes and even entire living cells using peptide elution under physiological conditions.

Differential organization of tonic and chronic B cell antigen receptors in the plasma membrane.

Stimulation of the B cell antigen receptor (BCR) triggers signaling pathways that promote the differentiation of B cells into plasma cells. Despite the pivotal function of BCR in B cell activation, the organization of the BCR on the surface of resting and antigen-activated B cells remains unclear. Here we show, using STED super-resolution microscopy, that IgM-containing BCRs exist predominantly as monomers and dimers in the plasma membrane of resting B cells, but form higher oligomeric clusters upon stimulation. By contrast, a chronic lymphocytic leukemia-derived BCR forms dimers and oligomers in the absence of a stimulus, but a single amino acid exchange reverts its organization to monomers in unstimulated B cells. Our super-resolution microscopy approach for quantitatively analyzing cell surface proteins may thus help reveal the nanoscale organization of immunoreceptors in various cell types.

Nanobody Detection of Standard Fluorescent Proteins Enables Multi-Target DNA-PAINT with High Resolution and Minimal Displacement Errors.

DNA point accumulation for imaging in nanoscale topography (PAINT) is a rapidly developing fluorescence super-resolution technique, which allows for reaching spatial resolutions below 10 nm. It also enables the imaging of multiple targets in the same sample. However, using DNA-PAINT to observe cellular structures at such resolution remains challenging. Antibodies, which are commonly used for this purpose, lead to a displacement between the target protein and the reporting fluorophore of 20⁻25 nm, thus limiting the resolving power. Here, we used nanobodies to minimize this linkage error to ~4 nm. We demonstrate multiplexed imaging by using three nanobodies, each able to bind to a different family of fluorescent proteins. We couple the nanobodies with single DNA strands via a straight forward and stoichiometric chemical conjugation. Additionally, we built a versatile computer-controlled microfluidic setup to enable multiplexed DNA-PAINT in an efficient manner. As a proof of principle, we labeled and imaged proteins on mitochondria, the Golgi apparatus, and chromatin. We obtained super-resolved images of the three targets with 20 nm resolution, and within only 35 minutes acquisition time.

Tryptophan-2,3-dioxygenase (TDO) inhibition ameliorates neurodegeneration by modulation of kynurenine pathway metabolites.

Metabolites of the kynurenine pathway (KP) of tryptophan (TRP) degradation have been closely linked to the pathogenesis of several neurodegenerative disorders. Recent work has highlighted the therapeutic potential of inhibiting two critical regulatory enzymes in this pathway-kynurenine-3-monooxygenase (KMO) and tryptophan-2,3-dioxygenase (TDO). Much evidence indicates that the efficacy of KMO inhibition arises from normalizing an imbalance between neurotoxic [3-hydroxykynurenine (3-HK); quinolinic acid (QUIN)] and neuroprotective [kynurenic acid (KYNA)] KP metabolites. However, it is not clear if TDO inhibition is protective via a similar mechanism or if this is instead due to increased levels of TRP-the substrate of TDO. Here, we find that increased levels of KYNA relative to 3-HK are likely central to the protection conferred by TDO inhibition in a fruit fly model of Huntington's disease and that TRP treatment strongly reduces neurodegeneration by shifting KP flux toward KYNA synthesis. In fly models of Alzheimer's and Parkinson's disease, we provide genetic evidence that inhibition of TDO or KMO improves locomotor performance and ameliorates shortened life span, as well as reducing neurodegeneration in Alzheimer's model flies. Critically, we find that treatment with a chemical TDO inhibitor is robustly protective in these models. Consequently, our work strongly supports targeting of the KP as a potential treatment strategy for several major neurodegenerative disorders and suggests that alterations in the levels of neuroactive KP metabolites could underlie several therapeutic benefits.