Biology subjects

Ganzinger, K. A.

Publications and source records attributed to Ganzinger, K. A..

12 recordsLinked to original sources

bioRxiv2026

Low CAR affinity enhances T-cell synapse dynamics and activation

Low affinity chimeric antigen receptors (CARs) can clinically outperform high-affinity variants, yet the molecular mechanisms underlying affinity-dependent activity remain unclear. Here, we show that low CAR affinity enables faster CAR cluster motion, which is central to T-cell activation. We compared low-affinity CAT and high-affinity FMC63 CARs signaling at different stages of activation on supported lipid bilayers functionalized with defined CD19 densities. At low CAR expression on dense CD19 bilayers, low-affinity CAT CARs formed more mature and dynamic synapses, recruited ZAP70 more efficiently, and showed faster microcluster motion. These proximal signaling changes were accompanied by stronger calcium flux and increased NFAT nuclear translocation. Our data support a model in which weaker, lower-affinity CAR-antigen interactions promote receptor turnover, microcluster mobility, and signaling-adaptor recruitment, thereby enhancing downstream signal propagation. Our work provides a mechanistic basis for the superior functional performance reported for affinity-tuned CD19 CAR-T cells.

biophysics↗

bioRxiv2026

Bilayer acoustic force spectroscopy (BAFS) for quantifying receptor-antigen binding strength in immune synapses

Immune cell receptor - ligand interactions are key to cancer immunotherapy. However, receptor-ligand affinities often fail to predict T-cell mediated cancer killing, while immune-target cell binding strength measurements are limited by low precision and high non-specific binding. Here we present bilayer acoustic force spectroscopy (BAFS), a method to quantify the binding strength of receptors in immune synapses that virtually eliminates non-specific binding and increases the resolving power by up to 50-fold. By replacing target cells with a supported lipid bilayer functionalized with antigens, BAFS avoids antigen-independent interactions and target cell heterogeneity, while maintaining the spatial self-organization of receptors that typifies active immune synapses. We demonstrate the high sensitivity and control by showing how CAR T-cell synapse strength depends on CD19 antigen density, and by revealing that CD8 synergistically strengthens {beta}TCR-pMHC synapses independently of Lck recruitment to CD8. BAFS is a general method that can be used broadly in immunotherapy screening and to dissect the complex molecular interactions that underpin immune synapse activation.

biophysics↗

bioRxiv2023

Uncovering the hidden mechanisms of giant unilamellar vesicle formation in cDICE

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield, while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE) that make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real-time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating both larger droplets and, likely, GUV-sized satellite droplets. According to existing theoretical models, the oil-water interface should allow for crossing of all droplets, but based on our observations and theoretical modelling of the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE, but ultimately also aid the development of more reliable and efficient methods for GUV production. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/562183v2_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@9764cforg.highwire.dtl.DTLVardef@15b63c7org.highwire.dtl.DTLVardef@f39e83org.highwire.dtl.DTLVardef@2bb63a_HPS_FORMAT_FIGEXP M_FIG C_FIG We developed a high-speed microscopy setup to study giant unilamellar vesicle formation in cDICE, revealing a complex droplet formation process occurring at the capillary orifice and size-selectivity at the oil-water interface.

synthetic biology↗

bioRxiv2023

Comparing kinetic proofreading and kinetic segregation for T cell receptor activation

The T cell receptor (TCR) is a key component of the adaptive immune system, recognizing foreign antigens and triggering an immune response. Competing models exist to explain the high sensitivity and selectivity of the TCR in discriminating self from non-self antigens, particularly models using kinetic proofreading (KP), kinetic segregation (KS), and combinations of the two. In this paper, we consider the role and importance of KS in TCR activation, using two models: classic KP (cKP), without KS, where antigen-TCR binding is required for activation, and a combination of KP and KS (KS-KP), where only residence within a close contact is required for activation. Building on previous work, our computational model is the first to permit a head-to-head comparison of these models in silico. While we find that both models can be used to explain the probability of TCR activation across much of the parameter space, we find biologically important regions in the parameter space where significant differences in performance can be expected. Furthermore, we show that the available experimental evidence may favour the KS-KP model over cKP. Our results may be used to motivate and guide future experiments to determine highly accurate computational models for the TCR. Author summaryThe T cell receptor (TCR) is a master of reliable sensing: it detects faint signals (rare ligands derived from foreign proteins) over high noise (abundant ligands derived from the bodys own proteins) to set T cells on a course to exterminate pathogens and tumours, a process that is central to our immune response. Despite decades of studying TCR signalling, we still do not know how the TCR can be so exceptionally sensitive and accurate. It is widely believed that kinetic proofreading (KP), in which the TCR binds to an antigen and triggers a series of phosphorylation steps prior to activation, plays an important role. However, recent results suggest that kinetic segregation (KS), in which binding is not required, is also important. These models are mutually exclusive, and yet both appear to explain various aspects of T cell activation. Our work directly addresses this puzzle. We develop a computational modeling framework which can simulate TCR activation by both KP-based and KS-based models, making it possible to compare them in silico for the first time. Using this framework, we find conditions under which the two models provide different responses, and we show that the limited experimental evidence to date is consistent with KS, which should motivate further investigation.

immunology↗

bioRxiv2022

The K2: Open-source simultaneous triple-color TIRF microscope for live-cell and single-molecule imaging

Imaging the dynamics and interactions of biomolecules at the single-molecule level in live cells and reconstituted systems has generated unprecedented knowledge about the biomolecular processes underlying many cellular functions. To achieve the speed and sensitivity needed to detect and follow individual molecules, these experiments typically require custom-built microscopes or custom modifications of commercial systems. The costs of such single-molecule microscopes, their technical complexity and the lack of open-source documentation on how to build custom setups therefore limit the accessibility of single-molecule imaging techniques. To advance the adaptation of dynamic single-molecule imaging by a wider community, we present the "K2": an open-source, simultaneous triple-color total internal reflection fluorescence (TIRF) microscope specifically designed for live-cell and single-molecule imaging. We explain our design considerations and provide step-by-step building instructions, parts list and full CAD models. The flexible design of this TIRF microscope allows users to customize it to their scientific and financial needs, or to re-use parts of our design to improve the capabilities of their existing setups without necessarily having to build a full copy of the K2 microscope.

biophysics↗

bioRxiv2022

DNA-PAINT single-particle tracking (DNA-PAINT-SPT) enables extended single-molecule studies of membrane protein interactions

DNA-PAINT based single-particle tracking (DNA-PAINT-SPT) has recently significantly enhanced observation times in in vitro SPT experiments by overcoming the constraints of fluorophore photobleaching. However, with the reported implementation, only a single target can be imaged and the technique cannot be applied straight to live cell imaging. Here we report on leveraging this technique from a proof-of-principle implementation to a useful tool for the SPT community by introducing simultaneous live cell dual-colour DNA-PAINT-SPT for quantifying protein dimerisation and tracking proteins in living cell membranes, demonstrating its improved performance over single-dye SPT.

biophysics↗

bioRxiv2021

Optimized cDICE for efficient reconstitution of biological systems in giant unilamellar vesicles

Giant unilamellar vesicles (GUVs) are often used to mimic biological membranes in reconstitution experiments. They are also widely used in research on synthetic cells as they provide a mechanically responsive reaction compartment that allows for controlled exchange of reactants with the environment. However, while many methods exist to encapsulate functional biomolecules in GUVs, there is no one-size-fits-all solution and reliable GUV fabrication still remains a major experimental hurdle in the field. Here, we show that defect-free GUVs containing complex biochemical systems can be generated by optimizing a double-emulsion method for GUV formation called continuous droplet interface crossing encapsulation (cDICE). By tightly controlling environmental conditions and tuning the lipid-in-oil dispersion, we show that it is possible to significantly improve the reproducibility of high-quality GUV formation as well as the encapsulation efficiency. We demonstrate efficient encapsulation for a range of minimal systems including a minimal actin cytoskeleton, membrane-anchored DNA nanostructures, and a functional PURE (Protein synthesis Using Recombinant Elements) system. Our optimized cDICE method displays promising potential to become a standard method in biophysics and bottom-up synthetic biology.

synthetic biology↗

bioRxiv2020

Tracking Single Particles for Hours via Continuous DNA-mediated Fluorophore Exchange

Fluorophores are commonly used to covalently label biomolecules for monitoring their motion in single particle tracking experiments. However, photobleaching is still a major bottleneck in these experiments, as the fluorophores finite photon budget typically limits observation times to merely a few seconds. Here, we overcome this inherent constraint via continuous fluorophore exchange based on DNA-PAINT, whereby fluorescently-labeled oligonucleotides bind to a 54 bp single-stranded DNA handle attached to the molecule of interest. When we assayed our approach in vitro by tracking single DNA origami, first surface-immobilized and subsequently diffusing on supported lipid bilayers, we were able to observe these origami for up to hours without losing their fluorescence signals. Our versatile and easily implemented labeling approach allows monitoring single-molecule motion and interactions over unprecedented observation periods, opening the doors to advanced quantitative studies.

biophysics↗

bioRxiv2020

ATP driven diffusiophoresis: active cargo transport without motor proteins

Morphogenesis and homeostasis of biological systems are intricately linked to gradient formation through energy dissipation. Such spatial organization may be achieved via reaction-diffusion or directional cargo transport, as prominently executed by motor proteins. In contrast to these processes that rely on specific protein interactions, active transport based on a non-specific, purely physical mechanism remains poorly explored. Here, by a joint experimental and theoretical approach, we describe a hidden function of the MinDE protein system from E. coli: Besides forming dynamic patterns, this system accomplishes the active transport of large, functionally unrelated cargo on membranes in vitro. Remarkably, this mechanism allows to sort diffusive objects according to their effective size, as evidenced using modular DNA origami-streptavidin nanostructures. We show that the diffusive fluxes of MinDE and cargo couple via density-dependent friction. This non-specific process constitutes a Maxwell-Stefan diffusiophoresis, so far undescribed in a biologically relevant setting. Such nonlinear coupling between diffusive fluxes could represent a generic physical mechanism for the intracellular organization of biomolecules.

biophysics↗

bioRxiv2019

Microfluidic trapping of vesicles reveals membrane-tension dependent FtsZ cytoskeletal re-organisation

The geometry of reaction compartments can affect the outcome of chemical reactions. Synthetic biology commonly uses giant unilamellar vesicles (GUVs) to generate cell-sized, membrane-bound reaction compartments. However, these liposomes are always spherical due to surface area minimization. Here, we have developed a microfluidic chip to trap and reversibly deform GUVs into rod- or cigar-like shapes, including a constriction site in the trap mimicking the membrane furrow in cell division. When we introduce into these GUVs the bacterial tubulin homologue FtsZ, the primary protein of the bacterial Z ring, we find that FtsZ organization changes from dynamic rings to elongated filaments upon GUV deformation, and that these FtsZ filaments align preferentially with the short GUV axis, in particular at the membrane neck. In contrast, pulsing Min oscillations in GUVs remained largely unaffected. We conclude that microfluidic traps are a useful tool for deforming GUVs into non-spherical membrane shapes, akin to those seen in cell division, and for investigating the effect of confinement geometry on biochemical reactions, such as protein filament self-organization.

synthetic biology↗

bioRxiv2018

Myosin-II activity generates a dynamic steady state with continuous actin turnover in a minimal actin cortex

Dynamic reorganization of the actomyosin cytoskeleton allows a fine-tuning of cell shape that is vital to many cellular functions. It is well established that myosin-II motors generate the forces required for remodeling the cell surface by imparting contractility to actin networks. An additional, less understood, role of myosin-II in cytoskeletal dynamics is believed to be in the regulation of actin turnover; it has been proposed that myosin activity increases actin turnover in various cellular contexts, presumably by contributing to disassembly. In vitro reconstitution of actomyosin networks has confirmed the role of myosin in actin network disassembly, but factors such as diffusional constraints and the use of stabilized filaments have thus far limited the observation of myosin-assisted actin turnover in these networks. Here, we present the reconstitution of a minimal dynamic actin cortex where actin polymerization is catalyzed on the membrane in the presence of myosin-II activity. We demonstrate that myosin activity leads to disassembly and redistribution in this simplified cortex. Consequently, a new dynamic steady state emerges in which actin filaments undergo constant turnover. Our findings suggest a multi-faceted role of myosin-II in fast remodeling of the eukaryotic actin cortex.

biophysics↗

bioRxiv2017

Constraining CD45 exclusion at close-contacts provides a mechanism for discriminatory T-cell receptor signalling

The T-cell receptor (TCR) triggers the elimination of pathogens and tumors by T lymphocytes. In order for this to avoid damage to the host, the receptor has to discriminate between thousands of peptide ligands presented by each host cell. Exactly how the TCR does this is unknown. In resting T-cells, the TCR is largely unphosphorylated due to the dominance of phosphatases over kinases expressed at the cell surface. When agonist peptides are presented to the TCR by major histocompatibility complex (MHC) proteins expressed by antigen-presenting cells (APCs), very fast receptor triggering occurs, leading to TCR phosphorylation. 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 develop and test a quantitative treatment of receptor triggering reliant only upon TCR dwell-time in phosphatase-depleted cell-cell contacts constrained in area by cell topography. Using the model and experimentally-derived parameters, we find that ligand discrimination is possible but that it depends crucially on individual contacts being 400 nm in diameter or smaller, i.e. the size generated by microvilli. The model not only correctly predicts the relative signaling potencies of known agonists and non-agonists, but achieves this in the absence of conventional, multi-step kinetic proof-reading. 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 crucially influences signaling outcomes.\n\nSignificance statementOne approach to testing biological theories is to determine if they are predictive. A simple, theoretical treatment of TCR triggering suggests that ligand discrimination by the receptor relies on just two physical principles: (1) the time TCRs spend in cell-cell contacts depleted of large tyrosine phosphatases; and (2) constraints on contact size imposed by T cells using finger-like protrusions to interrogate their targets. The theory not only allows agonistic and non-agonistic TCR ligands to be distinguished but predicts the relative signalling potencies of agonists with remarkable accuracy. This suggests that the theory captures the essential features of receptor triggering.

immunology↗