Biology subjects

Sutherland, D. S.

Publications and source records attributed to Sutherland, D. S..

9 recordsLinked to original sources

bioRxiv2026

Thymic Treg-derived T follicular regulatory cells prevent catastrophic humoral autoimmunity in response to TLR7-driven inflammation

T follicular regulatory cells (TFR) are a follicle-resident subset of regulatory T cells (TREG) that limit germinal center (GC) responses and enforce humoral tolerance. Although TFR are known to focus antibody responses to foreign antigens, how GC-specific regulation is maintained during inflammation remains unclear. Here, TLR7-driven inflammation unmasked a critical, non-redundant role for TFR in preserving immune tolerance. Selective loss of TFR caused severe autoimmunity and increased mortality, driven by inflammation-induced expansion of autoreactive B and T cell clones and epitope spreading. Autoreactivity resolved upon cessation of inflammation in TFR-sucicient mice but persisted in their absence. Mechanistically, TFR limited the establishment and expansion of spontaneous GCs in response to inflammation, and responding TFR displayed transcriptional, phenotypic, and clonal features of thymic TREG. This suggests that whereas TFH upregulate FoxP3 to shut down end-stage GCs, it is thymic TREG-derived TFR that safeguard GC integrity under inflammatory stress to prevent lethal autoimmunity.

immunology↗

bioRxiv2025

Display of clustered antigen by follicular dendritic cells tunes B-cell receptor activation

To generate high-affinity antibodies against pathogens, B cells engage antigens displayed on the surface of follicular dendritic cells (FDCs) in secondary lymphoid tissues. Binding of cognate antigen by the B-cell receptor (BCR) causes Src kinase activation and phosphorylation of SYK (pSYK), serving as a platform for downstream signaling. However, how presentation of antigen by FDCs influence antigen binding and intracellular signaling in B cells remains unclear. Here, we show that antigen occurs in submicron pre-clusters on FDCs. We use nanoscale ligand patterning to present clustered antigens on tension force tethers in relevant cluster geometries in vitro. Clustering of antigens greatly increases BCR activation in a pattern-size-dependent manner, correlating to exclusion of the tyrosine phosphatase CD45 from BCR-antigen complexes. Increased height of antigen placement reduces CD45 exclusion with concomitant reduction in pSYK. Super-resolution imaging reveals reduced levels of pSYK at the edges of antigen patterns where CD45 interactions are greater, indicating a geometric control of BCR triggering.

immunology↗

bioRxiv2025

Nanoscale precise stamping of biomolecule patterns using DNA origami

Understanding the importance of ligand patterning in biological processes requires precise control over molecular positioning and spacing. While DNA origami structures offer nanoscale precision in biomolecule arrangement, their biological applications are limited by challenges related to their structural stability, scalability, and surface area. Here, we present a straightforward and rapid DNA origami stamping technique for transferring nanoscale oligonucleotide patterns onto surfaces, visualized using DNA-PAINT super-resolution microscopy to quantitatively assess the stamping efficiency and precision across different stamp types. Unlike traditional top-down methods that require specialized equipment, our technique provides an accessible, self-assembled platform for surface patterning, with versatility across various substrates via modifiable pattern-transfer oligonucleotides. We demonstrate reliable, efficient, and precise pattern transfer at single-molecule resolution, enabling new opportunities to study distance-dependent biological processes, including receptor activation, multivalent binding, and enzymatic cascades across broader spatial scales and different detection techniques. The use of the passivated surface limits non-specific interactions with unpatterned areas and enables control over the interaction between the biological target and the patterned biomolecules. Our method advances surface patterning by combining DNA nanotechnology with single-molecule imaging techniques, expanding access to cost-effective analytical approaches and potentially enabling multiplexed detection and live measurements.

bioengineering↗

bioRxiv2025

Familial Alzheimer disease mutation identifies novel role of SORLA in release of neurotrophic exosomes

Sortilin-related receptor with A-type repeats (SORLA) is an intracellular sorting receptor that directs target proteins between endocytic and secretory compartments of cells. Mutations in SORL1, encoding SORLA, are common in individuals suffering from Alzheimer disease (AD) of unknown etiology. Conceptually, characterization of inheritable SORL1 variants associated with AD can provide important new information about functions of this receptor relevant for aging brain health. Here, we focused on elucidation of the AD-associated variant SORLA N1358S, carrying a mutation in the main ligand binding domain of the receptor. Using unbiased quantitative proteome screens, we identified major alterations in the mutant receptor interactome linked to biogenesis and secretion of exosomes. Using advanced biophysical, cell biological, as well as functional studies in stem cell-derived human cell models we corroborated impaired release and loss of neurotrophic action of exosomes from neurons and microglia expressing SORLAN1358S. An impaired neurotrophic potential was attributed to an altered exosomal content of RNA binding proteins and associated microRNAs, known to control neuronal growth and maturation. Our studies identified a so far unknown function for SORLA in controlling the quantity and trophic quality of extracellular vesicles secreted by cells, and they argue for impaired cellular cross talk through exosomes as a pathological trail contributing to the risk of AD seen with carriers of SORL1 variants.

neuroscience↗

bioRxiv2025

Serial intravital microscopy reveals temporal dynamics of autoreactive germinal centers in the spleen

The spleen plays a key role in clearing blood-borne infections and is involved in autoimmune and hematological disorders. It undergoes extensive remodeling during inflammation and immune reactions, but its localization in the peritoneal cavity has hampered studies of these dynamic changes. Here, we establish and validate a protocol for serial 2-photon microscopy of the murine spleen to capture dynamic processes in the living animal. As a proof-of-principle, we elucidate the expansion and contraction of autoreactive germinal centers (GCs) induced by epicutaneous application of the small-molecule TLR7 agonist resiquimod (R848). Leveraging a biocompatible abdominal imaging window, intravital labeling techniques, and fluorescent reporters, we follow GCs up to 180 {micro}m below the capsule for more than 2 weeks by tracking follicular dendritic cell (FDC) networks. This was accomplished without appreciable perturbation of normal physiology, paving the way for a deeper understanding of the biology of the spleen and its associated disease states. HighlightAn abdominal imaging window allowing the study of dynamic processes in the spleen of live mice over the course of several weeks.

immunology↗

bioRxiv2023

Kinetics and dynamics of single-molecule multivalent interactions revealed by plasmon-enhanced fluorescence

Multivalency as an interaction principle is widely utilized in nature. It enables specific and strong binding by multiple weak interactions through enhanced avidity and is a core process in immune recognition and cellular signaling and a current concept in drug design. Rapid binding and unbinding of monovalent constituent interactions during multivalent binding creates dynamics that require a single-molecule approach to be studied. Here, we use the high signals from plasmon enhanced fluorescence of nanoparticles to extract binding kinetics and dynamics of multivalent interactions on the single-molecule level and in real-time. We study mono-, bi-and trivalent binding interactions using a DNA Holliday Junction as a model construct with programmable valency. Furthermore, we introduce a model framework for binding kinetics that involves the binding restriction during multivalent interactions to take into account the structural conformation of multivalent molecules allowing quantitative comparison. We used this approach to explore how length and flexibility of the DNA ligands affect binding restriction and binding strength, where overall binding strength decreased with spacer length. For trivalent systems increasing spacer length was found to activate binding in the trivalent state giving insight into the design of multivalent drug or targeting moieties. Interestingly we could exploit the rapidly decaying near fields of the plasmon that induce a strong dependence of the signal to position of the fluorophore to observe binding dynamics during single multivalent binding events.

biophysics↗

bioRxiv2023

Microplate Format Protein Nanopatterning for High-Throughput Screening of Cellular Microenvironments

An advanced protein nanopatterned cell culture platform is engineered to emulate the extracellular matrixs complexity, enabling precise nanoscale biomolecule copatterning to mimic environments analogous to native tissue for cellular assays. Nanopatterns fabricated through sparse colloidal lithography, with 100 nm to 800 nm features in separate wells, are seamlessly integrated into standard microplate formats (96-well/384-well). Robust patterns are built from fully PEGylated, passivated thin glass coverslips optimized for minimal nonspecific interactions. Biotin-avidin binding and click chemistry to ensure the accurate and robust localization of bioligands. The transparent, metal-free substrates are free of topographical interference, rendering them ideal for diverse fluorescence microscopy techniques encompassing single-molecule TIRFM and extensive high-throughput imaging. The structural stability of these nanopatterns persists beyond a year in storage and long-term in cell culture conditions, endorsing their application for prolonged experimental studies and potential for widespread academic and industrial use. The platform has been demonstrated for nanopatterning an array of biomolecules, from small molecules to proteins, DNA, and extracellular matrix components, instrumental for studying cell signaling. Experiments with C2C12 cells demonstrated the exceptional specificity of the nanopatterned microplates, with nonspecific adhesion remaining below 2% and the platforms ability to elicit size-dependent cellular reactions when interfaced with nanopatterned fibronectin.

bioengineering↗

bioRxiv2020

Tracing the in vivo fate of nanoparticles with a "non-self" biological identity

ABSTRACTNanoparticles can acquire a biomolecular corona with a species-specific biological identity. However, “non-self” incompatibility of recipient biological systems is often not considered, for example, when rodents are used as a model organism for preclinical studies of biomolecule-inspired nanomedicines. Using zebrafish embryos as an emerging model for nano-bioimaging, here we unraveled the in vivo fate of intravenously injected 70 nm SiO2 nanoparticles with a protein corona pre-formed from fetal bovine serum (FBS), representing a non-self biological identity. Strikingly rapid sequestration and endolysosomal acidification of nanoparticles with the pre-formed FBS corona were observed in scavenger endothelial cells within minutes after injection. This led to loss of blood vessel integrity and inflammatory activation of macrophages over the course of several hours. As unmodified nanoparticles or the equivalent dose of FBS proteins alone failed to induce the observed pathophysiology, this signifies how the corona enriched with a differential repertoire of proteins can determine the fate of the nanoparticles in vivo. Our findings thus reveal the adverse outcome triggered by incompatible protein coronas and indicate a potential pitfall in the use of mismatched species combinations during nanomedicine development.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2020

Mapping and identification of soft corona proteins at nanoparticles and their impact on cellular association

The current understanding of the biological identity that nanoparticles may acquire in a given biological milieu is mostly inferred from the hard component of the protein corona (HC). The composition of soft corona (SC) proteins and their biological relevance have remained elusive due to the lack of analytical separation methods. Here, we identified a set of specific corona proteins with weak interactions at silica and polystyrene nanoparticles by using an in situ click-chemistry reaction. We show that these SC proteins are present also in the HC, but are specifically enriched after the capture, suggesting that the main distinction between HC and SC is the differential binding strength of the same proteins. Interestingly, the weakly interacting proteins in the SC are revealed as modulators of nanoparticle-cell association, in spite of their short residence time. We therefore highlight that weak interactions of proteins at nanoparticles should be considered when evaluating nano-bio interfaces.

pharmacology and toxicology↗