Biology subjects

Brameshuber, M.

Publications and source records attributed to Brameshuber, M..

6 recordsLinked to original sources

bioRxiv2026

A Minimally Perturbative DARPin Probe for Quantitative Fluorescence Imaging of the Human TCR-CD3 Complex

Fluorescence microscopy is a powerful tool for dissecting the molecular mechanisms of T-cell antigen recognition in living cells, but its quantitative insight critically depends on non-perturbative, high-quality probes. Here, we repurpose a small (~15 kDa) CD3epsilon-binding DARPin (designed ankyrin repeat proteins) to a fluorescent label for T-cell receptor (TCR)/CD3 complexes on primary human CD8+ T-cells, with the aim of generating a powerful tool for quantitative analysis, single-molecule tracking, and advanced imaging of TCR dynamics. We show that the DARPin binds CD3{varepsilon} with high affinity and selectivity and using single molecule tracking and brightness analysis, we characterize the TCR-CD3 diffusion behavior and show that the DARPin binds to both CD3epsilon; subunits. Importantly, labeling preserves antigen sensitivity: on supported lipid bilayers presenting cognate pMHC, T-cells remain responsive, assemble synapses, form TCR microclusters, and initiate signaling similar to unlabeled controls. We further demonstrate compatibility with lattice light-sheet microscopy for volumetric imaging of T-cell - APC interactions in living cells. Together, these results establish DARPins as versatile, minimally perturbative probes for high resolution, quantitative studies of T cell synapse organization and signaling.

biophysics↗

bioRxiv2023

Monomeric agonist peptide/MHCII complexes activate T-cells in an autonomous fashion

Molecular crowding of agonist peptide/MHC class II complexes (pMHCIIs) with structurally similar, yet per se non-stimulatory endogenous pMHCIIs has been postulated to sensitize T-cells for the recognition of single antigens on the surface of dendritic cells and B-cells. When testing this premise with the use of advanced live cell microscopy, we observed pMHCIIs as monomeric, randomly distributed entities diffusing rapidly after entering the APC surface. Synaptic TCR-engagement of highly abundant endogenous pMHCIIs was low or non-existent and affected neither TCR-engagement of rare agonist pMHCII in early and advanced synapses nor agonist-induced TCR-proximal signaling. Our findings highlight the capacity of single freely diffusing agonist pMHCIIs to elicit the full T-cell response in an autonomous and peptide-specific fashion with consequences for adaptive immunity and immunotherapeutic approaches. SHORT SUMMARYPlatzer et al. revealed via highly quantitative and single molecule live cell microscopy the nature of peptide-loaded MHC class II molecules (pMHCII) as monomeric, densely populating, randomly distributed and predominantly rapidly diffusing entities on the surface of B-cells and dendritic cells. Low abundant stimulatory agonist pMHCII acted as autonomous units with the highest chance of T-cell detection when equally spread on APCs. The presence of bystander-pMHCII previously termed "co-agonist pMHC" affected neither synaptic agonist -TCR-binding nor efficiencies of T-cell recognition. "Co-agonist"-TCR-binding resembled random molecular collisions. Findings inform the design of T-cell-based immunotherapies.

immunology↗

bioRxiv2021

3D single molecule localization microscopy reveals the topography of the immunological synapse at isotropic precision below 15 nm

T-cells engage with antigen-presenting cells in search for antigenic peptides and form transient interfaces termed immunological synapses. A variety of protein-protein interactions in trans-configuration defines the topography of the synapse and orchestrates the antigen-recognition process. In turn, the synapse topography affects receptor binding rates and the mutual segregation of proteins due to size exclusion effects. For better understanding it is hence critical to map the 3D topography of the immunological synapse at high precision. Current methods, however, provide only rather coarse images of the protein distribution within the synapse, which do not reach the dimension of the protein ectodomains. Here, we applied supercritical angle fluorescence microscopy combined with defocused imaging, which allows 3-dimensional single molecule localization microscopy (3D-SMLM) at an isotropic localization precision below 15 nm. Experiments were performed on hybrid synapses between primary T-cells and functionalized glass-supported lipid bilayers. We used 3D-SMLM to quantify the cleft size within the synapse by mapping the position of the T-cell receptor (TCR) with respect to the supported lipid bilayer, yielding average distances of 18 nm up to 31 nm for activating and non-activating bilayers, respectively.

biophysics↗

bioRxiv2020

DNA origami demonstrate the unique stimulatory power of single pMHCs as T-cell antigens

T-cells detect with their T-cell antigen receptors (TCRs) the presence of rare peptide/MHC complexes (pMHCs) on the surface of antigen presenting cells (APCs). How they convert a biochemical interaction into a signaling response is poorly understood, yet indirect evidence pointed to the spatial antigen arrangement on the APC surface as a critical factor. To examine this, we engineered a biomimetic interface based on laterally mobile functionalized DNA origami platforms, which allow for nanoscale control over ligand distances without interfering with the cell-intrinsic dynamics of receptor clustering. We found that the minimum signaling unit required for efficient T-cell activation consisted of two ligated TCRs within a distance of 20 nanometers, if TCRs were stably engaged by monovalent antibody fragments. In contrast, antigenic pMHCs stimulated T-cells robustly as well-isolated entities. These results identify the minimal requirements for effective TCR-triggering and validate the exceptional stimulatory potency of transiently engaging pMHCs.

biophysics↗

bioRxiv2019

Verifying molecular clusters by 2-color localization microscopy and significance testing

While single-molecule localization microscopy (SMLM) offers the invaluable prospect to visualize cellular structures below the diffraction limit of light microscopy, its potential could not be fully capitalized due to its inherent susceptibility to blinking artifacts. Particularly, overcounting of single molecule localizations has impeded a reliable and sensitive detection of biomolecular nanoclusters. Here we introduce a 2-Color Localization microscopy And Significance Testing Approach (2-CLASTA), providing a parameter-free statistical framework for the analysis of SMLM data via significance testing methods. 2-CLASTA yields p-values for the null hypothesis of random biomolecular distributions, independent of the blinking behavior of the chosen fluorescent labels. We validated the method both by computer simulations as well as experimentally, using protein concatemers as a mimicry of biomolecular clustering. As the new approach it is not affected by overcounting artifacts, it is able to detect biomolecular clustering of various shapes at high sensitivity down to a level of dimers.

biophysics↗

bioRxiv2019

Unscrambling Fluorophore Blinking for Comprehensive Cluster Detection via PALM

Determining nanoscale protein distribution via Photoactivated Localization Microscopy (PALM) mandates precise knowledge of the applied fluorophores blinking properties to counteract overcounting artifacts that distort the resulting biomolecular distributions. Here, we present a readily applicable methodology to determine, optimize and quantitatively account for the blinking behavior of any PALM-compatible fluorophore. Using a custom-designed platform we revealed complex blinking of two photoswitchable fluorescence proteins (PS-CFP2 and mEOS3.2) and two photoactivatable organic fluorophores (PA Janelia Fluor 549 and Abberior CAGE 635) with blinking cycles on time scales of several seconds. Incorporating such detailed information in our simulation-based analysis package allowed for robust evaluation of molecular clustering based on individually recorded single molecule localization maps. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=45 SRC="FIGDIR/small/545152v5_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@b0930org.highwire.dtl.DTLVardef@883897org.highwire.dtl.DTLVardef@13b7304org.highwire.dtl.DTLVardef@1fc136c_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗