Antibody agonists trigger immune receptor signaling through local exclusion of receptor-type protein tyrosine phosphatases.

Antibodies can block immune receptor engagement or trigger the receptor machinery to initiate signaling. We hypothesized that antibody agonists trigger signaling by sterically excluding large receptor-type protein tyrosine phosphatases (RPTPs) such as CD45 from sites of receptor engagement. An agonist targeting the costimulatory receptor CD28 produced signals that depended on antibody immobilization and were sensitive to the sizes of the receptor, the RPTPs, and the antibody itself. Although both the agonist and a non-agonistic anti-CD28 antibody locally excluded CD45, the agonistic antibody was more effective. An anti-PD-1 antibody that bound membrane proximally excluded CD45, triggered Src homology 2 domain-containing phosphatase 2 recruitment, and suppressed systemic lupus erythematosus and delayed-type hypersensitivity in experimental models. Paradoxically, nivolumab and pembrolizumab, anti-PD-1-blocking antibodies used clinically, also excluded CD45 and were agonistic in certain settings. Reducing these agonistic effects using antibody engineering improved PD-1 blockade. These findings establish a framework for developing new and improved therapies for autoimmunity and cancer.

Initiation of T cell signaling by CD45 segregation at 'close contacts'.

It has been proposed that the local segregation of kinases and the tyrosine phosphatase CD45 underpins T cell antigen receptor (TCR) triggering, but how such segregation occurs and whether it can initiate signaling is unclear. Using structural and biophysical analysis, we show that the extracellular region of CD45 is rigid and extends beyond the distance spanned by TCR-ligand complexes, implying that sites of TCR-ligand engagement would sterically exclude CD45. We also show that the formation of 'close contacts', new structures characterized by spontaneous CD45 and kinase segregation at the submicron-scale, initiates signaling even when TCR ligands are absent. Our work reveals the structural basis for, and the potent signaling effects of, local CD45 and kinase segregation. TCR ligands have the potential to heighten signaling simply by holding receptors in close contacts.

Single-Molecule Stoichiometry of Supramolecular Complexes.

The use of single-molecule microscopy is introduced as a method to quantify the photophysical properties of supramolecular complexes rapidly at ultra low concentrations (<1 nM), previously inaccessible. Using a model supramolecular system based on the host-guest complexation of cucurbit[n]uril (CB[n]) macrocycles together with a fluorescent guest (Ant910Me), we probe fluorescent CB[n] host-guest complexes in the single molecule regime. We show quantification and differentiation of host-guest photophysics and stoichiometries, both in aqueous media and noninvasively in hydrogel, by thresholding detected photons. This methodology has wide reaching implications in aiding the design of next-generation materials with programmed and controlled properties.

vLUME: 3D virtual reality for single-molecule localization microscopy.

vLUME is a virtual reality software package designed to render large three-dimensional single-molecule localization microscopy datasets. vLUME features include visualization, segmentation, bespoke analysis of complex local geometries and exporting features. vLUME can perform complex analysis on real three-dimensional biological samples that would otherwise be impossible by using regular flat-screen visualization programs.

3D structures of individual mammalian genomes studied by single-cell Hi-C.

The folding of genomic DNA from the beads-on-a-string-like structure of nucleosomes into higher-order assemblies is crucially linked to nuclear processes. Here we calculate 3D structures of entire mammalian genomes using data from a new chromosome conformation capture procedure that allows us to first image and then process single cells. The technique enables genome folding to be examined at a scale of less than 100 kb, and chromosome structures to be validated. The structures of individual topological-associated domains and loops vary substantially from cell to cell. By contrast, A and B compartments, lamina-associated domains and active enhancers and promoters are organized in a consistent way on a genome-wide basis in every cell, suggesting that they could drive chromosome and genome folding. By studying genes regulated by pluripotency factor and nucleosome remodelling deacetylase (NuRD), we illustrate how the determination of single-cell genome structure provides a new approach for investigating biological processes.

Rational design of a conformation-specific antibody for the quantification of Aß oligomers.

Protein misfolding and aggregation is the hallmark of numerous human disorders, including Alzheimer's disease. This process involves the formation of transient and heterogeneous soluble oligomers, some of which are highly cytotoxic. A major challenge for the development of effective diagnostic and therapeutic tools is thus the detection and quantification of these elusive oligomers. Here, to address this problem, we develop a two-step rational design method for the discovery of oligomer-specific antibodies. The first step consists of an "antigen scanning" phase in which an initial panel of antibodies is designed to bind different epitopes covering the entire sequence of a target protein. This procedure enables the determination through in vitro assays of the regions exposed in the oligomers but not in the fibrillar deposits. The second step involves an "epitope mining" phase, in which a second panel of antibodies is designed to specifically target the regions identified during the scanning step. We illustrate this method in the case of the amyloid ß (Aß) peptide, whose oligomers are associated with Alzheimer's disease. Our results show that this approach enables the accurate detection and quantification of Aß oligomers in vitro, and in Caenorhabditis elegans and mouse hippocampal tissues.

Wild-type sTREM2 blocks Aß aggregation and neurotoxicity, but the Alzheimer's R47H mutant increases Aß aggregation.

TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aß and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aß oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aß oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aß oligomerization and disaggregated preformed Aß oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aß fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aß-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aß but rather promotes Aß protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aß-induced release of sTREM2, which blocks Aß aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aß aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.

A cell topography-based mechanism for ligand discrimination by the T cell receptor.

The T cell receptor (TCR) initiates the elimination of pathogens and tumors by T cells. To avoid damage to the host, the receptor must be capable of discriminating between wild-type and mutated self and nonself peptide ligands presented by host cells. Exactly how the TCR does this is unknown. In resting T cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over the kinases expressed at the cell surface. However, when agonist peptides are presented to the TCR by major histocompatibility complex proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering, i.e., TCR phosphorylation, occurs. Recent work suggests that this depends on the local exclusion of the phosphatases from regions of contact of the T cells with the APCs. Here, we developed and tested a quantitative treatment of receptor triggering reliant only on TCR dwell time in phosphatase-depleted cell contacts constrained in area by cell topography. Using the model and experimentally derived parameters, we found that ligand discrimination likely depends crucially on individual contacts being ∼200 nm in radius, matching the dimensions of the surface protrusions used by T cells to interrogate their targets. The model not only correctly predicted the relative signaling potencies of known agonists and nonagonists but also achieved this in the absence of kinetic proofreading. Our work provides a simple, quantitative, and predictive molecular framework for understanding why TCR triggering is so selective and fast and reveals that, for some receptors, cell topography likely influences signaling outcomes.

resPAINT: Accelerating Volumetric Super-Resolution Localisation Microscopy by Active Control of Probe Emission.

Points for accumulation in nanoscale topography (PAINT) allows practically unlimited measurements in localisation microscopy but is limited by background fluorescence at high probe concentrations, especially in volumetric imaging. We present reservoir-PAINT (resPAINT), which combines PAINT and active control of probe photophysics. In resPAINT, an activatable probe "reservoir" accumulates on target, enabling a 50-fold increase in localisation rate versus conventional PAINT, without compromising contrast. By combining resPAINT with large depth-of-field microscopy, we demonstrate super-resolution imaging of entire cell surfaces. We generalise the approach by implementing various switching strategies and 3D imaging techniques. Finally, we use resPAINT with a Fab to image membrane proteins, extending the operating regime of PAINT to include a wider range of biological interactions.

The Costs of Close Contacts: Visualizing the Energy Landscape of Cell Contacts at the Nanoscale.

Cell-cell contacts often underpin signaling between cells. For immunology, the binding of a T cell receptor to an antigen-presenting pMHC initiates downstream signaling and an immune response. Although this contact is mediated by proteins on both cells creating interfaces with gap sizes typically around 14 nm, many, often contradictory observations have been made regarding the influence of the contact on parameters such as the binding kinetics, spatial distribution, and diffusion of signaling proteins within the contact. Understanding the basic physical constraints on probes inside this crowded environment will help inform studies on binding kinetics and dynamics of signaling of relevant proteins in the synapse. By tracking quantum dots of different dimensions for extended periods of time, we have shown that it is possible to obtain the probability of a molecule entering the contact, the change in its diffusion upon entry, and the impact of spatial heterogeneity of adhesion protein density in the contact. By analyzing the contacts formed by a T cell interacting with adhesion proteins anchored to a supported lipid bilayer, we find that probes are excluded from contact entry in a size-dependent manner for gap-to-probe differences of 4.1 nm. We also observed probes being trapped inside the contact and a decrease in diffusion of up to 85% in dense adhesion protein contacts. This approach provides new, to our knowledge, insights into the nature of cell-cell contacts, revealing that cell contacts are highly heterogeneous because of topography- and protein-density-related processes. These effects are likely to profoundly influence signaling between cells.

Mapping Surface Hydrophobicity of α-Synuclein Oligomers at the Nanoscale.

Proteins fold into a single structural ensemble but can also misfold into many diverse structures including small aggregates and fibrils, which differ in their toxicity. The aggregate surface properties play an important role in how they interact with the plasma membrane and cellular organelles, potentially inducing cellular toxicity, however, these properties have not been measured to date due to the lack of suitable methods. Here, we used a spectrally resolved, super-resolution imaging method combined with an environmentally sensitive fluorescent dye to measure the surface hydrophobicity of individual aggregates formed by the protein α-synuclein (αS), whose aggregation is associated with Parkinson's disease. We show that the surface of soluble oligomers is more hydrophobic than fibrils and populates a diverse range of coexisting states. Overall, our data show that the conversion of oligomers to fibril-like aggregates and ultimately to fibrils results in a reduction in both hydrophobicity and the variation in hydrophobicity. This funneling characteristic of the energy landscape explains many of the observed properties of αS aggregates and may be a common feature of aggregating proteins.

Single-Molecule Light-Sheet Imaging of Suspended T Cells.

Adaptive immune responses are initiated by triggering of the T cell receptor. Single-molecule imaging based on total internal reflection fluorescence microscopy at coverslip/basal cell interfaces is commonly used to study this process. These experiments have suggested, unexpectedly, that the diffusional behavior and organization of signaling proteins and receptors may be constrained before activation. However, it is unclear to what extent the molecular behavior and cell state is affected by the imaging conditions, i.e., by the presence of a supporting surface. In this study, we implemented single-molecule light-sheet microscopy, which enables single receptors to be directly visualized at any plane in a cell to study protein dynamics and organization in live, resting T cells. The light sheet enabled the acquisition of high-quality single-molecule fluorescence images that were comparable to those of total internal reflection fluorescence microscopy. By comparing the apical and basal surfaces of surface-contacting T cells using single-molecule light-sheet microscopy, we found that most coated-glass surfaces and supported lipid bilayers profoundly affected the diffusion of membrane proteins (T cell receptor and CD45) and that all the surfaces induced calcium influx to various degrees. Our results suggest that, when studying resting T cells, surfaces are best avoided, which we achieve here by suspending cells in agarose.

Nanoscopic Characterisation of Individual Endogenous Protein Aggregates in Human Neuronal Cells.

The aberrant misfolding and subsequent conversion of monomeric protein into amyloid aggregates characterises many neurodegenerative disorders, including Parkinson's and Alzheimer's diseases. These aggregates are highly heterogeneous in structure, generally of low abundance and typically smaller than the diffraction limit of light (≈250 nm). To overcome the challenges these characteristics pose to the study of endogenous aggregates formed in cells, we have developed a method to characterise them at the nanometre scale without the need for a conjugated fluorophore. Using a combination of DNA PAINT and an amyloid-specific aptamer, we demonstrate that this technique is able to detect and super-resolve a range of aggregated species, including those formed by α-synuclein and amyloid-ß. Additionally, this method enables endogenous protein aggregates within cells to be characterised. We found that neuronal cells derived from patients with Parkinson's disease contain a larger number of protein aggregates than those from healthy controls.

Maximizing the field of view and accuracy in 3D Single Molecule Localization Microscopy.

Super-resolution techniques that localize single molecules in three dimensions through point spread function (PSF) engineering are very sensitive to aberrations and optical alignment. Here we show how double-helix point spread function is affected by such mis-alignment and aberration. Specifically, we demonstrate through simulation and experiment how misplacement of phase masks in infinity corrected systems is a common source of significant loss of accuracy. We also describe an optimal alignment and calibration procedure to correct for these errors. In combination, these optimizations allow for a maximal field of view with high accuracy and precision. Though discussed with reference to double-helix point spread function (DHPSF), the optimization techniques are equally applicable to other engineered PSFs.

Nanobodies raised against monomeric ɑ-synuclein inhibit fibril formation and destabilize toxic oligomeric species.

BACKGROUND: The aggregation of the protein ɑ-synuclein (ɑS) underlies a range of increasingly common neurodegenerative disorders including Parkinson's disease. One widely explored therapeutic strategy for these conditions is the use of antibodies to target aggregated ɑS, although a detailed molecular-level mechanism of the action of such species remains elusive. Here, we characterize ɑS aggregation in vitro in the presence of two ɑS-specific single-domain antibodies (nanobodies), NbSyn2 and NbSyn87, which bind to the highly accessible C-terminal region of ɑS. RESULTS: We show that both nanobodies inhibit the formation of ɑS fibrils. Furthermore, using single-molecule fluorescence techniques, we demonstrate that nanobody binding promotes a rapid conformational conversion from more stable oligomers to less stable oligomers of ɑS, leading to a dramatic reduction in oligomer-induced cellular toxicity. CONCLUSIONS: The results indicate a novel mechanism by which diseases associated with protein aggregation can be inhibited, and suggest that NbSyn2 and NbSyn87 could have significant therapeutic potential.

Optical Structural Analysis of Individual α-Synuclein Oligomers.

Small aggregates of misfolded proteins play a key role in neurodegenerative disorders. Such species have proved difficult to study due to the lack of suitable methods capable of resolving these heterogeneous aggregates, which are smaller than the optical diffraction limit. We demonstrate here an all-optical fluorescence microscopy method to characterise the structure of individual protein aggregates based on the fluorescence anisotropy of dyes such as thioflavin-T, and show that this technology is capable of studying oligomers in human biofluids such as cerebrospinal fluid. We first investigated in vitro the structural changes in individual oligomers formed during the aggregation of recombinant α-synuclein. By studying the diffraction-limited aggregates we directly evaluated their structural conversion and correlated this with the potential of aggregates to disrupt lipid bilayers. We finally characterised the structural features of aggregates present in cerebrospinal fluid of Parkinson's disease patients and age-matched healthy controls.

Single-Molecule Imaging of Wnt3A Protein Diffusion on Living Cell Membranes.

Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport.

Three-Dimensional Super-Resolution in Eukaryotic Cells Using the Double-Helix Point Spread Function.

Single-molecule localization microscopy, typically based on total internal reflection illumination, has taken our understanding of protein organization and dynamics in cells beyond the diffraction limit. However, biological systems exist in a complicated three-dimensional environment, which has required the development of new techniques, including the double-helix point spread function (DHPSF), to accurately visualize biological processes. The application of the DHPSF approach has so far been limited to the study of relatively small prokaryotic cells. By matching the refractive index of the objective lens immersion liquid to that of the sample media, we demonstrate DHPSF imaging of up to 15-µm-thick whole eukaryotic cell volumes in three to five imaging planes. We illustrate the capabilities of the DHPSF by exploring large-scale membrane reorganization in human T cells after receptor triggering, and by using single-particle tracking to image several mammalian proteins, including membrane, cytoplasmic, and nuclear proteins in T cells and embryonic stem cells.

Ultrasensitive Measurement of Ca2+ Influx into Lipid Vesicles Induced by Protein Aggregates.

To quantify and characterize the potentially toxic protein aggregates associated with neurodegenerative diseases, a high-throughput assay based on measuring the extent of aggregate-induced Ca2+ entry into individual lipid vesicles has been developed. This approach was implemented by tethering vesicles containing a Ca2+ sensitive fluorescent dye to a passivated surface and measuring changes in the fluorescence as a result of membrane disruption using total internal reflection microscopy. Picomolar concentrations of Aß42 oligomers could be observed to induce Ca2+ influx, which could be inhibited by the addition of a naturally occurring chaperone and a nanobody designed to bind to the Aß peptide. We show that the assay can be used to study aggregates from other proteins, such as α-synuclein, and to probe the effects of complex biofluids, such as cerebrospinal fluid, and thus has wide applicability.