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.

Protein nanobarcodes enable single-step multiplexed fluorescence imaging.

Multiplexed cellular imaging typically relies on the sequential application of detection probes, as antibodies or DNA barcodes, which is complex and time-consuming. To address this, we developed here protein nanobarcodes, composed of combinations of epitopes recognized by specific sets of nanobodies. The nanobarcodes are read in a single imaging step, relying on nanobodies conjugated to distinct fluorophores, which enables a precise analysis of large numbers of protein combinations. Fluorescence images from nanobarcodes were used as input images for a deep neural network, which was able to identify proteins with high precision. We thus present an efficient and straightforward protein identification method, which is applicable to relatively complex biological assays. We demonstrate this by a multicell competition assay, in which we successfully used our nanobarcoded proteins together with neurexin and neuroligin isoforms, thereby testing the preferred binding combinations of multiple isoforms, in parallel.

Actinin-4 controls survival signaling in B cells by limiting the lateral mobility of B-cell antigen receptors.

The structure and dynamics of F-actin networks in the cortical area of B cells control the signal efficiency of B-cell antigen receptors (BCRs). Although antigen-induced signaling has been studied extensively, the role of cortical F-actin in antigen-independent tonic BCR signaling is less well understood. Because these signals are essential for the survival of B cells and are consequently exploited by several B-cell lymphomas, we assessed how the cortical F-actin structure influences tonic BCR signal transduction. We employed genetic variants of a primary cell-like B-cell line that can be rendered quiescent to show that cross-linking of actin filaments by α-actinin-4 (ACTN4), but not ACTN1, is required to preserve the dense architecture of F-actin in the cortical area of B cells. The reduced cortical F-actin density in the absence of ACTN4 resulted in increased lateral BCR diffusion. Surprisingly, this was associated with reduced tonic activation of BCR-proximal effector proteins, extracellular signal-regulated kinase, and pro-survival pathways. Accordingly, ACTN4-deficient B-cell lines and primary human B cells exhibit augmented apoptosis. Hence, our findings reveal that cortical F-actin architecture regulates antigen-independent tonic BCR survival signals in human B cells.

Protein target highlights in CASP15: Analysis of models by structure providers.

We present an in-depth analysis of selected CASP15 targets, focusing on their biological and functional significance. The authors of the structures identify and discuss key protein features and evaluate how effectively these aspects were captured in the submitted predictions. While the overall ability to predict three-dimensional protein structures continues to impress, reproducing uncommon features not previously observed in experimental structures is still a challenge. Furthermore, instances with conformational flexibility and large multimeric complexes highlight the need for novel scoring strategies to better emphasize biologically relevant structural regions. Looking ahead, closer integration of computational and experimental techniques will play a key role in determining the next challenges to be unraveled in the field of structural molecular biology.

Simple and Highly Efficient Detection of PSD95 Using a Nanobody and Its Recombinant Heavy-Chain Antibody Derivatives.

The post-synaptic density protein 95 (PSD95) is a crucial scaffolding protein participating in the organization and regulation of synapses. PSD95 interacts with numerous molecules, including neurotransmitter receptors and ion channels. The functional dysregulation of PSD95 as well as its abundance and localization has been implicated with several neurological disorders, making it an attractive target for developing strategies able to monitor PSD95 accurately for diagnostics and therapeutics. This study characterizes a novel camelid single-domain antibody (nanobody) that binds strongly and with high specificity to rat, mouse, and human PSD95. This nanobody allows for more precise detection and quantification of PSD95 in various biological samples. We expect that the flexibility and unique performance of this thoroughly characterized affinity tool will help to further understand the role of PSD95 in normal and diseased neuronal synapses.

A fluorescent probe for STED microscopy to study NIP-specific B cells.

We have developed a series of monovalent fluorophore-conjugated affinity probes based on the hapten 3-nitro-4-hydroxy-5-iodophenylacetyl (NIP), which is widely used as a model antigen to study B lymphocytes and the functional principles of B cell antigen receptors (BCRs). We successfully used them in flow-cytometry, confocal and super-resolution microscopy techniques.

Exotic nuclear spin behavior in dendritic macromolecules.

Dendrimers are a class of branched, highly symmetric macromolecules that have been shown to be useful for a vast number of different applications. Potential uses as fluorescence sensors, in catalysis and perhaps most importantly in medical applications as drug delivery systems or cytotoxica have been proposed. Herein we report on an exotic behaviour of the nuclear spins in a dendritic macromolecule in the presence of different paramagnetic ions. We show that the stability of the long lived nuclear singlet state, is affected by the presence of Cu(II), whereas other ions did not have any influence at all. This effect could not be observed in the case of a simple tripeptide, in which the nuclear singlet stability was influenced by all investigated paramagnetic ions, a potentially useful effect in the development of Cu(II) selective probes. By adding a fluorescent marker to our molecule we could show that the nuclear singlet multimer (NUSIMER) is taken up by living cells. Furthermore we were able to show that nuclear singlet state NMR can be used to investigate the NUSIMER in the presence of living cells, showing that an application in in vivo NMR can be feasible.

Quantum Defects as a Toolbox for the Covalent Functionalization of Carbon Nanotubes with Peptides and Proteins.

Single-walled carbon nanotubes (SWCNTs) are a 1D nanomaterial that shows fluorescence in the near-infrared (NIR, >800 nm). In the past, covalent chemistry was less explored to functionalize SWCNTs as it impairs NIR emission. However, certain sp3 defects (quantum defects) in the carbon lattice have emerged that preserve NIR fluorescence and even introduce a new, red-shifted emission peak. Here, we report on quantum defects, introduced using light-driven diazonium chemistry, that serve as anchor points for peptides and proteins. We show that maleimide anchors allow conjugation of cysteine-containing proteins such as a GFP-binding nanobody. In addition, an Fmoc-protected phenylalanine defect serves as a starting point for conjugation of visible fluorophores to create multicolor SWCNTs and in situ peptide synthesis directly on the nanotube. Therefore, these quantum defects are a versatile platform to tailor both the nanotube's photophysical properties as well as their surface chemistry.

The extracellular membrane-proximal domain of membrane-bound IgE restricts B cell activation by limiting B cell antigen receptor surface expression.

Immunoglobulin E (IgE) antibodies are key mediators of allergic reactions. Due to their potentially harmful anaphylactic properties, their production is tightly regulated. The membrane-bound isoform of IgE (mIgE), which is an integral component of the B cell antigen receptor, has been shown to be critical for the regulation of IgE responses in mice. In primate species including humans, mIgE can be expressed in two isoforms that are produced by alternative splicing of the primary ε Ig heavy chain transcript, and differ in the absence or presence of an extracellular membrane-proximal domain (EMPD) consisting of 52 amino acids. However, the function of the EMPD remains unclear. Here, we demonstrate that the EMPD restricts surface expression of mIgE-containing BCRs in human and murine B cells. The EMPD does not interfere with BCR assembly but acts as an autonomous endoplasmic reticulum retention domain. Limited surface expression of EMPD-containing mIgE-BCRs caused impaired activation of intracellular signaling cascades and hence represents a regulatory mechanism that may control the production of potentially anaphylactic IgE antibodies in primate species.

Nanobody-Conjugated Nanotubes for Targeted Near-Infrared In Vivo Imaging and Sensing.

Fluorescent nanomaterials such as single-walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP-binding nanobodies to DNA-wrapped SWCNTs. This approach combines the targeting capabilities of GFP-binding nanobodies and the nonbleaching near-infrared fluorescence (850-1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin-5-GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime.

Boron-Containing Probes for Non-optical High-Resolution Imaging of Biological Samples.

Boron has been employed in materials science as a marker for imaging specific structures by electron energy loss spectroscopy (EELS) or secondary ion mass spectrometry (SIMS). It has a strong potential in biological analyses as well; however, the specific coupling of a sufficient number of boron atoms to a biological structure has proven challenging. Herein, we synthesize tags containing closo-1,2-dicarbadodecaborane, coupled to soluble peptides, which were integrated in specific proteins by click chemistry in mammalian cells and were also coupled to nanobodies for use in immunocytochemistry experiments. The tags were fully functional in biological samples, as demonstrated by nanoSIMS imaging of cell cultures. The boron signal revealed the protein of interest, while other SIMS channels were used for imaging different positive ions, such as the cellular metal ions. This allows, for the first time, the simultaneous imaging of such ions with a protein of interest and will enable new biological applications in the SIMS field.

Super-resolution imaging for cell biologists: concepts, applications, current challenges and developments.

The recent 2014 Nobel Prize in chemistry honored an era of discoveries and technical advancements in the field of super-resolution microscopy. However, the applications of diffraction-unlimited imaging in biology have a long road ahead and persistently engage scientists with new challenges. Some of the bottlenecks that restrain the dissemination of super-resolution techniques are tangible, and include the limited performance of affinity probes and the yet not capillary diffusion of imaging setups. Likewise, super-resolution microscopy has introduced new paradigms in the design of projects that require imaging with nanometer-resolution and in the interpretation of biological images. Besides structural or morphological characterization, super-resolution imaging is quickly expanding towards interaction mapping, multiple target detection and live imaging. Here we review the recent progress of biologists employing super-resolution imaging, some pitfalls, implications and new trends, with the purpose of animating the field and spurring future developments.

Tools and limitations to study the molecular composition of synapses by fluorescence microscopy.

The synapse is densely packed with proteins involved in various highly regulated processes. Synaptic protein copy numbers and their stoichiometric distribution have a drastic influence on neuronal integrity and function. Therefore, the molecular analysis of synapses is a key element to understand their architecture and function. The overall structure of the synapse has been revealed with an exquisite amount of details by electron microscopy. However, the molecular composition and the localization of proteins are more easily addressed with fluorescence imaging, especially with the improved resolution achieved by super-resolution microscopy techniques. Notably, the fast improvement of imaging instruments has not been reflected in the optimization of biological sample preparation. During recent years, large efforts have been made to generate affinity probes smaller than conventional antibodies adapted for fluorescent super-resolution imaging. In this review, we briefly discuss the current views on synaptic organization and necessary key technologies to progress in the understanding of synaptic physiology. We also highlight the challenges faced by current fluorescent super-resolution methods, and we describe the prerequisites for an ideal study of synaptic organization.

Secondary-ion mass spectrometry of genetically encoded targets.

Secondary ion mass spectrometry (SIMS) is generally used in imaging the isotopic composition of various materials. It is becoming increasingly popular in biology, especially for investigations of cellular metabolism. However, individual proteins are difficult to identify in SIMS, which limits the ability of this technology to study individual compartments or protein complexes. We present a method for specific protein isotopic and fluorescence labeling (SPILL), based on a novel click reaction with isotopic probes. Using this method, we added (19) F-enriched labels to different proteins, and visualized them by NanoSIMS and fluorescence microscopy. The (19) F signal allowed the precise visualization of the protein of interest, with minimal background, and enabled correlative studies of protein distribution and cellular metabolism or composition. SPILL can be applied to biological systems suitable for click chemistry, which include most cell-culture systems, as well as small model organisms.

Advancing cell biology with nanoscale fluorescence imaging: essential practical considerations.

Recently, biologists have gained access to several far-field fluorescence nanoscopy (FN) technologies that allow the observation of cellular components with ~20 nm resolution. FN is revolutionizing cell biology by enabling the visualization of previously inaccessible subcellular details. While technological advances in microscopy are critical to the field, optimal sample preparation and labeling are equally important and often overlooked in FN experiments. In this review, we provide an overview of the methodological and experimental factors that must be considered when performing FN. We present key concepts related to the selection of affinity-based labels, dyes, multiplexing, live cell imaging approaches, and quantitative microscopy. Consideration of these factors greatly enhances the effectiveness of FN, making it an exquisite tool for numerous biological applications.

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.

Pre-fibrillar alpha-synuclein variants with impaired beta-structure increase neurotoxicity in Parkinson's disease models.

The relation of alpha-synuclein (alphaS) aggregation to Parkinson's disease (PD) has long been recognized, but the mechanism of toxicity, the pathogenic species and its molecular properties are yet to be identified. To obtain insight into the function different aggregated alphaS species have in neurotoxicity in vivo, we generated alphaS variants by a structure-based rational design. Biophysical analysis revealed that the alphaS mutants have a reduced fibrillization propensity, but form increased amounts of soluble oligomers. To assess their biological response in vivo, we studied the effects of the biophysically defined pre-fibrillar alphaS mutants after expression in tissue culture cells, in mammalian neurons and in PD model organisms, such as Caenorhabditis elegans and Drosophila melanogaster. The results show a striking correlation between alphaS aggregates with impaired beta-structure, neuronal toxicity and behavioural defects, and they establish a tight link between the biophysical properties of multimeric alphaS species and their in vivo function.

Endosomal sorting of readily releasable synaptic vesicles.

Neurotransmitter release is achieved through the fusion of synaptic vesicles with the neuronal plasma membrane (exocytosis). Vesicles are then retrieved from the plasma membrane (endocytosis). It was hypothesized more than 3 decades ago that endosomes participate in vesicle recycling, constituting a slow endocytosis pathway required especially after prolonged stimulation. This recycling model predicts that newly endocytosed vesicles fuse with an endosome, which sorts (organizes) the molecules and buds exocytosis-competent vesicles. We analyzed here the endosome function using hippocampal neurons, isolated nerve terminals (synaptosomes), and PC12 cells by stimulated emission depletion microscopy, photooxidation EM, and several conventional microscopy assays. Surprisingly, we found that endosomal sorting is a rapid pathway, which appeared to be involved in the recycling of the initial vesicles to be released on stimulation, the readily releasable pool. In agreement with the endosomal model, the vesicle composition changed after endocytosis, with the newly formed vesicles being enriched in plasma membrane proteins. Vesicle proteins were organized in clusters both in the plasma membrane (on exocytosis) and in the endosome. In the latter compartment, they segregated from plasma membrane components in a process that is likely important for sorting/budding of newly developed vesicles from the endosome.

Trafficking proteins show limited differences in mobility across different postsynaptic spines.

The function of the postsynaptic compartment is based on the presence and activity of postsynaptic receptors, whose dynamics are controlled by numerous scaffolding, signaling and trafficking proteins. Although the receptors and the scaffolding proteins have received substantial attention, the trafficking proteins have not been investigated extensively. Their mobility rates are unknown, and it is unclear how the postsynaptic environment affects their dynamics. To address this, we analyzed several trafficking proteins (α-synuclein, amphiphysin, calmodulin, doc2a, dynamin, and endophilin), estimating their movement rates in the dendritic shaft, as well as in morphologically distinct "mushroom" and "stubby" postsynapse types. The diffusion parameters were surprisingly similar across dendritic compartments, and a few differences between proteins became evident only in the presence of a synapse neck. We conclude that the movement of trafficking proteins is not strongly affected by the postsynaptic compartment, in stark contrast to the presynapse, which regulates strongly the movement of such proteins.

Heat denaturation enables multicolor X10-STED microscopy.

Expansion microscopy (ExM) improves imaging quality by physically enlarging the biological specimens. In principle, combining a large expansion factor with optical super-resolution should provide extremely high imaging precision. However, large expansion factors imply that the expanded specimens are dim and are therefore poorly suited for optical super-resolution. To solve this problem, we present a protocol that ensures the expansion of the samples up to 10-fold, in a single expansion step, through high-temperature homogenization (X10ht). The resulting gels exhibit a higher fluorescence intensity than gels homogenized using enzymatic digestion (based on proteinase K). This enables the sample analysis by multicolor stimulated emission depletion (STED) microscopy, for a final resolution of 6-8 nm in neuronal cell cultures or isolated vesicles. X10ht also enables the expansion of 100-200 µm thick brain samples, up to 6-fold. The better epitope preservation also enables the use of nanobodies as labeling probes and the implementation of post-expansion signal amplification. We conclude that X10ht is a promising tool for nanoscale resolution in biological samples.