Biology subjects

Klotzsch, E.

Publications and source records attributed to Klotzsch, E..

7 recordsLinked to original sources

bioRxiv2026

Aquaporin-4-Specific T Cell Responses in NMOSD Revealed by mRNA-Engineered Dendritic Cells

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease of the central nervous system characterized by loss of immune tolerance to the water channel aquaporin-4 (AQP4). Current therapies do not specifically restore AQP4 tolerance or selectively suppress antigen-specific immune responses. Reprogramming patients own dendritic cells (DCs) with mRNA to induce tolerance represents a promising therapeutic strategy. A key prerequisite for this approach is generating mRNAs encoding disease-relevant autoantigens recognized by the patients immune system. Here, we generated recombinant mRNAs encoding human AQP4 and evaluated T cell responses to autologous DCs transfected with AQP4 mRNA in eight NMOSD patients and ten healthy controls. Transfected DCs were co-cultured with autologous T cells and stimulated twice. The assay detected robust AQP4-specific T cell response in a patient with recent disease activity who was not receiving immunosuppressive therapy, demonstrating its ability to identify clinically relevant autoreactive T cell responses. These findings establish the feasibility of an mRNA-based antigen-specific T cell assay for studying NMOSD pathogenesis, stratifying patients by T cell involvement, and supporting development of personalized tolerance-inducing therapies. Summary SentenceRecombinant mRNA encoding human aquaporin-4 (AQP4) was introduced into dendritic cells from NMOSD patients, enabling the detection of antigen-specific T cell responses, with the strongest activation observed in a patient with recent disease activity, highlighting the potential of this approach for immune monitoring and for understanding the role of T cells in the pathogenesis of NMOSD.

immunology↗

bioRxiv2025

Guided maturation of human neuromuscular organoids via electrical stimulation

Organoids derived from human pluripotent stem cells (hPSCs) are emerging as powerful models for studying development and disease. Despite their physiological relevance, the predictive power of organoids remains limited by the immature state of the constituent cells, posing a major challenge for mechanistic studies of adult physiology and late-onset diseases and disorders. Here, we establish a strategy for enhancing the maturation status of human neuromuscular organoids (NMOs) through chronic Electrical Pulse Stimulation (EPS). We demonstrate that low-frequency EPS, applied early on during NMO development and maintained over several weeks, promotes structural and functional maturation of neuromuscular junctions (NMJs). Independent of stimulation waveform dynamics, EPS-trained NMOs (EPS-NMOs) displayed stronger and more frequent spontaneous contractions that persisted long after stimulation ceased. Quantitative imaging and transcriptomic analyses revealed a robust improvement in EPS-NMO skeletal muscle and neural tissue morphology, coordinated regulation of lineage-specific biomarkers, and upregulation of gene programmes associated with mature neuromuscular function. Mechanobiological measurements further demonstrated increased EPS-NMO tissue stiffness and faster relaxation dynamics, consistent with advanced excitation-contraction coupling and force generation. Collectively, these findings establish EPS as a powerful, non-invasive, and on-demand modality for driving the morphological and functional maturation of complex organoid systems.

bioengineering↗

bioRxiv2023

The prion-like domain of the chloroplast RNA binding protein CP29A is required for cold-induced phase separation next to nucleoids and supports RNA splicing and translation during cold acclimation

Arabidopsis thaliana is capable of producing photosynthetic tissue with active chloroplasts at temperatures as low as 4{degrees}C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif (RRM) domains of CP29A. The resulting droplets display liquid-like properties and are found in close proximity to chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role for plant cold resistance.

plant biology↗

bioRxiv2023

SPI-1 virulence gene expression modulates motility of Salmonella Typhimurium in a proton motive force- and adhesins-dependent manner

Both the bacterial flagellum and the evolutionary related injectisome encoded on the Salmonella pathogenicity island 1 (SPI-1) play crucial roles during the infection cycle of Salmonella species. The interplay of both is highlighted by the complex cross-regulation that includes transcriptional control of the flagellar master regulatory operon flhDC by HilD, the master regulator of SPI-1 gene expression. Contrary to the HilD-dependent activation of flagellar gene expression, we report here that activation of HilD resulted in a dramatic loss of motility, which was dependent on the presence of SPI-1. Single cell analyses revealed that HilD-activation results in a SPI-1-dependent induction of the stringent response and a pronounced decrease of proton motive force (PMF), while flagellation was not affected. We further found that activation of HilD enhanced the adhesion of Salmonella to epithelial cells. A transcriptome analysis revealed a concomitant upregulation of several adhesin systems, which when overproduced, phenocopied the HilD-induced motility defect. We propose that a combination of SPI-1-dependent depletion of the PMF and upregulation of adhesins upon HilD-activation allows flagellated Salmonella to rapidly modulate their motility during infection, thereby enabling efficient adhesion to host cells and delivery of effector proteins.

microbiology↗

bioRxiv2021

Nanoconfinement of Microvilli Alters Gene Expression and Boosts T cell Activation

T cells sense and respond to their local environment at the nanoscale by forming small actin-rich protrusions, called microvilli, which play critical roles in signaling and antigen recognition, particularly at the interface with the antigen presenting cells. However, the mechanisms by which microvilli contribute to cell signaling and activation is largely unknown. Here, we present a tunable engineered system that promotes microvilli formation and T cell signaling via physical stimuli. We discovered that nanoporous surfaces favored microvilli formation, and markedly altered gene expression in T cells and promoted their activation. Mechanistically, confinement of microvilli inside of nanopores leads to size-dependent sorting of membrane-anchored proteins, specifically segregating CD45 phosphatases and T cell receptors (TCR) from the tip of the protrusions when microvilli are confined in 200 nm pores, but not in 400 nm pores. Consequently, formation of TCR nanoclustered hotspots within 200 nm pores, allows sustained and augmented signaling that prompts T cell activation even in the absence of TCR agonists. The synergistic combination of mechanical and biochemical signals on porous surfaces presents a straightforward strategy to investigate the role of microvilli in T cell signaling as well as to boost T cell activation and expansion for application in the growing field of adoptive immunotherapy.

bioengineering↗

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↗