Biology subjects

Schuetz, G. J.

Publications and source records attributed to Schuetz, G. J..

8 recordsLinked to original sources

bioRxiv2025

Refractive index mapping below the diffraction limit via single molecule localization microscopy

Single molecule localization microscopy (SMLM) is a powerful method to image biological samples in three dimensions below the diffraction limit of light microscopy. Beyond the position of the emitter, the shape of the single molecule point spread function provides additional information, for example about the refractive properties of the sample between the emitter and the glass coverslip. Here, we show that combination of SMLM with atomic force microscopy (AFM) allows to map the refractive index of a biological sample at sub-diffraction resolution and at a precision only limited by measurement errors of SMLM and AFM. We showcase the new method by the determination of the refractive index of isolated single collagen fibrils. Variabilities both in refractive index and the swelling behavior of single fibrils upon drying and rehydration exposed deviations from the ensemble behavior, demonstrating differential hydration of single collagen fibrils. Mapping the refractive index along single collagen fibrils revealed substantial fluctuations at characteristic length scales below 500 nm, which indicates structural heterogeneity of collagen fibrils at the length scale of single collagen molecules.

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↗

bioRxiv2022

Quantitation of FGFR3 signaling via GRB2 recruitment on micropatterned surfaces

Fibroblast growth factor receptors (FGFRs) initiate signal transduction via the RAS/MAPK pathway by their tyrosine-kinase activation known to determine cell-growth, tissue differentiation and apoptosis. Recently, many missense mutations have been reported for FGFR3, but we only know the functional effect for a handful of them. Some of these mutations result in aberrant FGFR3 signaling and are associated with various genetic disorders and oncogenic conditions. Here we employed micropatterned surfaces to specifically enrich fluorophore-tagged FGFR3 (mGFP-FGFR3) in certain areas of the plasma membrane of living cells. Receptor activation was then quantified via the recruitment of the downstream signal transducer GRB2 tagged with mScarlet (GRB2-mScarlet) to FGFR3 patterns. With this system, we tested the activation of FGFR3 upon ligand addition (fgf1 and fgf2) in the wildtype (WT), as well as in different FGFR3 mutants associated with congenital disorders (G380R, Y373C, K650Q, K650E). Our data showed that the addition of ligands increased GRB2 recruitment to WT FGFR3, with fgf1 having a stronger effect than fgf2. For all mutants, we found an increased basal receptor activity, and only for two of the four mutants (G380R and K650Q), activity was further increased upon ligand addition. Compared to previous reports, two mutant receptors (K650Q and K650E) had either an unexpectedly high or low activation state, respectively. This may be explained by the different receptor populations probed, since the micropatterning method specifically reports on signaling events at the plasma membrane. Graphical AbstractSpecifications: The maximum size of the image should be 200 x 500 pixels with a minimum resolution of 300 dpi, using Arial font with a size of 10-16 points; Preferred file types: TIFF, EPS, PDF or MS Office files O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=120 SRC="FIGDIR/small/487861v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@4a279eorg.highwire.dtl.DTLVardef@6f35d1org.highwire.dtl.DTLVardef@13661e7org.highwire.dtl.DTLVardef@155b480_HPS_FORMAT_FIGEXP M_FIG C_FIG Research HighlightsO_LIQuantification of FGFR3 signaling in live cells on micropatterned surfaces C_LIO_LIAnalysis of GRB2 recruitment to the mature receptor at the plasma membrane C_LIO_LILigand-independent kinase activation of FGFR3 mutants C_LIO_LIActivation of FGFR3 at the cell surface can be different than in bulk cell extracts C_LI

molecular biology↗

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↗

bioRxiv2021

Strategies for the site-specific decoration of DNA origami nanostructures with functionally intact proteins

DNA origami structures provide flexible scaffolds for the organization of single biomolecules with nanometer precision. While they find increasing use for a variety of biological applications, the functionalization with proteins at defined stoichiometry, high yield, and under preservation of protein function remains challenging. In this study, we applied single molecule fluorescence microscopy in combination with a cell biological functional assay to systematically evaluate different strategies for the site-specific decoration of DNA origami structures, focusing on efficiency, stoichiometry and protein functionality. Using an activating ligand of the T-cell receptor (TCR) as protein of interest, we found that two commonly used methodologies underperformed with regard to stoichiometry and protein functionality. While strategies employing tetravalent wildtype streptavidin for coupling of a biotinylated TCR-ligand yielded mixed populations of DNA origami structures featuring up to 3 proteins, the use of divalent (dSAv) or DNA-conjugated monovalent streptavidin (mSAv) allowed for site-specific attachment of a single biotinylated TCR-ligand. The most straightforward decoration strategy, via covalent DNA conjugation, resulted in a 3-fold decrease in ligand potency, likely due to charge-mediated impairment of protein function. Replacing DNA with charge-neutral PNA in a ligand conjugate emerged as the coupling strategy with the best overall performance in our study, as it produced the highest yield with no multivalent DNA origami structures and fully retained protein functionality. With our study we aim to provide guidelines for the stoichiometrically defined, site-specific functionalization of DNA origami structures with proteins of choice serving a wide range of biological applications.

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↗