Biology subjects

Schuetz, G.

Publications and source records attributed to Schuetz, G..

5 recordsLinked to original sources

bioRxiv2026

A workflow for the identification of oligomeric structures on tilted sample planes in Cryo-SMLM

We introduce a workflow to identify oligomeric structures that are recorded with single-molecule localization microscopy (SMLM) under cryogenic conditions. Typically, these oligomers are assumed to consist of protomers arranged as equilateral two-dimensional polygons and every protomer is labeled with a dye molecule for visualization. Unlike previous work, we consider scenarios in which the sample plane has an unknown orientation relative to the focal plane. Our contribution is a high-precision plane-fitting algorithm to determine the sample plane, combined with geometrical transformations and two circle-fitting algorithms to identify the oligomeric structures. Our simulations on synthetic data demonstrate that the proposed workflow achieves high accuracy in estimating both the unknown tilted plane and the oligomer size.

biophysics↗

bioRxiv2023

Single-molecule FRET and tracking of transfected biomolecules: multi-dimensional protein dynamics in living cells

Proteins and DNA in cells exhibit different conformational states, which are influenced by dynamic interactions with other biomolecules. All these interactions are affected by the molecules localization within the cell, i.e., their compartmentalization. Such, in cellula, compartment-specific dynamics is difficult to measure, because of limitations in instrumentation, autofluorescence of cells, and the necessity to track diffusing molecules. Here, we present a bottom-up engineering approach, which allows us to track transfected proteins in cellula and to analyze time-resolved single-molecule FRET efficiencies. This has been achieved by alternating laser excitation (ALEX) based three-channel (donor, acceptor and FRET) tracking with a live-cell HILO microscope. We validate our strategy by characterizing long-term static-FRET traces of customized DNA with known dye positions. We utilize two different transfection strategies, namely a biological (Streptolysin-O toxin protein) and a physical one (Microinjection). By comparing in vitro and in cellula measurements we show that the cellular environment in this case changes the FRET efficiency by about 25%. In addition, we evaluate single-molecule FRET traces for the heat shock protein Hsp90 in cellula. The obtained FRET efficiency distribution is largely consistent with known Hsp90 structures and in vitro distributions, but also shows some clear differences. Altogether, we show that FRET-TTB (Forster Resonance Energy Transfer-Tracking of Transfected Biomolecules) opens the path to study protein state changes of transfected biomolecules in cellula, including time-resolved cellular localization. SignificanceInside cells, proteins and DNA can change shape depending on their environment and their interactions. Studying these changes for single biomolecules is challenging because they move around in the cell and the cell environment makes it hard to see them clearly. Here, we demonstrate a new technique called FRET-TTB that enables us to track individual proteins inside living cells over time using single-molecule fluorescence microscopy. We validate the approach with custom DNA and apply it to study the heat shock protein Hsp90. Our results show that this method can reveal changes in protein conformation and location within living cells.

biophysics↗

bioRxiv2022

Activating mutations in FGFR3 are associated with clonal expansion events and high de novo rates in the male germline

Delayed fatherhood results in a higher risk to inherit a new germline mutation that might result in a congenital disorder in the offspring. In particular, some FGFR3 mutations increase in frequency with age, but there are still a large number of uncharacterized FGFR3 mutations that could be expanding in the male germline with potentially early or late-onset effects in the offspring. Here, we investigated the mutation frequency in the DNA of human testis and sperm and the activation state of the expressed mutant protein of eight different FGFR3 variants categorized by ClinVar as deleterious, benign, or not reported. Overall, the ligand-independent activation of the mutant protein resulted in a increased number of mutant sperm; although, strong activating mutations did not necessarily result in the highest frequencies. Moreover, only two mutants c.952G>A and c.1620C>A showed an increase with the donors age; the latter also forming larger clonal expansions in the testis. We also showed that the prediction of deleteriousness of a mutation is not always accurate, and similar in silico scores can reflect either a gain-of-function or loss-of-function. Our approach led to the discovery of two novel variants c.1261G>A and c.952G>A to have promiscuous FGFR3 activation and increased mutation frequencies in the male germline. The large fraction of donors with mutations suggests a high de novo rate potentially explained by a selective advantage before the maturation of the male germline. This sequence-function study provides important data for the evaluation and interpretation of variants with relevant clinical implications.

genetics↗

bioRxiv2020

Functionalized bead assay to measure 3-dimensional traction forces during T-cell activation

When T-cells probe their environment for antigens, the bond between the T-cell receptor (TCR) and the peptide-loaded major histocompatibility complex (MHC) is put under tension, thereby influencing the antigen discrimination process. Yet, the quantification of such forces in the context of T-cell signaling is technically challenging. Common approaches such as traction force microscopy (TFM) employ a global readout of the force fields, e.g. by measuring the displacements of hydrogel-embedded marker beads. Recent data, however, indicated that T-cells exert tensile forces locally via TCR-enriched microvilli while scanning the surface of antigen-presenting cells. Here, we developed a traction force microscopy platform, which allows for quantifying the pulls exerted via T-cell microvilli, in both tangential and normal directions, during T-cell activation. For this, we immobilized specific T-cell activating antibodies directly on the marker beads used to read out the hydrogel deformation. Microvilli targeted the functionalized beads, as confirmed by superresolution microscopy of the local actin organization. Moreover, we found that cellular components, such as actin, TCR and CD45 reorganize upon interaction with the beads, such that actin forms a vortex-like ring structure around the beads and TCR is enriched at the bead surface, whereas, CD45 is excluded from bead-microvilli contacts. Significance statementDuring the antigen recognition process, T-cells explore and probe their environment via microvilli, which exert local pushes and pulls at the surface of the antigen presenting cell. It is currently believed that these forces influence or even enable the antigen recognition process. Here, we describe the development of a platform, which allows us to quantify the magnitude and direction of traction forces exerted locally by T cell microvilli. Simultaneous Ca2+ imaging was used to link the measured forces to the overall T cell activation status. Superresolution microscopy resolved the contact sites of bead-microvilli contact at the nanoscale: cells contacted beads via actin vortex-like structures, which excluded the phosphatase CD45 from the contacts.

biophysics↗

bioRxiv2020

Temporal Analysis of T-Cell Receptor-Imposed Forces via Quantitative Single Molecule FRET Measurements

Mechanical forces acting on ligand-engaged T-cell receptors (TCRs) have previously been implicated in T-cell antigen recognition, yet their magnitude, spread, and temporal behavior are still poorly defined. We here report a FRET-based sensor equipped with a TCR-reactive single chain antibody fragment, which was tethered to planar supported lipid bilayers (SLBs) and informs most directly on the magnitude and kinetics of TCR-imposed forces at the single molecule level. When confronting T-cells with gel-phase SLBs we observed both prior and upon T-cell activation a single, well-resolvable force-peak of approximately 5 pN and force loading rates on the TCR of 1.5 pN per second. When facing fluid SLBs instead, T-cells still exerted tensile forces yet with threefold reduced magnitude and only prior to but not upon activation. Our findings do not only provide first truly molecular information on TCR-imposed forces within the immunological synapse, they also recalibrate their significance in antigen recognition.

biophysics↗