Biology subjects

Winter, L.

Publications and source records attributed to Winter, L..

8 recordsLinked to original sources

bioRxiv2026

Mechanochemical synchronization of force oscillations in connective tissue models

Cells in a tissue coordinate their behavior collectively by mechanical and chemical feedback with their surroundings. To study how such feedback mechanisms interact, we embed multicellular spheroids from mouse and primary human fibroblasts of different origin in 3D collagen matrices. Immediately after embedding, spheroids apply steadily increasing, radially symmetric, inward-directed forces to the surrounding matrix. Typically, within a few hours, collective force oscillations emerge with an average time interval of {approx}30 min. Similar force oscillations also arise in engineered, parallel-aligned microtissues attached to flexible pillars. Force oscillations propagate between neighboring cells as a mechanical wave, accompanied by calcium waves that travel at comparable speeds of several hundred m/min. Blocking gap junctions or mechanosensitive Piezo1 channels impairs these oscillations, indicating that calcium signaling contributes to their propagation. Their amplitude and frequency are regulated by integrin-mediated feedback in actin-rich protrusions and by ERK signaling, which also controls collective invasion into the surrounding matrix. Neighboring spheroids mechanically synchronize their force oscillations in phase and frequency over distances of up to {approx}2 mm without direct contact. Together, our results show that oscillatory mechanochemical feedback enables multicellular systems to coordinate force generation and invasive migration across cellular and millimeter length scales in 3D matrices.

biophysics↗

bioRxiv2026

A primary human muscle cell-based assay for detecting myasthenia gravis autoantibody binding and assessing AChR cluster impairment

Myasthenia gravis (MG) is an autoimmune disease caused by pathogenic autoantibodies against proteins at the neuromuscular junction (NMJ). The diagnosis and clinical management of MG patients largely relies on the detection of antigen-specific autoantibodies targeting acetylcholine receptor (AChR) or muscle-specific kinase (MuSK). Yet a subset of patients remains seronegative for known MG autoantibodies, highlighting a critical need for alternative approaches to identify pathogenic NMJ antibodies. We established a new human in vitro model of the NMJ based on primary human muscle cells that recapitulates key features of the NMJ: differentiation to myotubes, expression of key NMJ proteins and formation of postsynaptic AChR clusters in response to agrin stimulation. The model allows new insights into myogenesis and genetic muscle diseases, and the new muscle cell-based assay (CBA) detected autoantibodies in sera from patients with AChR- and MuSK-positive MG with 96.43% sensitivity and 100% specificity, while healthy control sera showed no reactivity. Incubation with patient sera significantly reduced AChR clustering compared to controls, demonstrating functional pathogenic effects. Thus, we established a physiologically relevant human NMJ model that enables detection and functional characterization of neuromuscular autoantibodies. This novel approach addresses a key limitation of current antigen-specific diagnostics and provides a method for improved detection and characterization of MG antibodies, independent of antigen specificity. One Sentence SummaryWe established a postsynaptic human in vitro neuromuscular junction model to assess binding and pathogenicity of MG autoantibodies. Key messagesO_ST_ABSWhat is already known on this topic?C_ST_ABSCurrent diagnosis of myasthenia gravis (MG) relies largely on the detection of antigen-specific autoantibodies against AChR and MuSK, leaving a clinically relevant subset of patients seronegative. What are the new findings?We established a physiologically relevant human in vitro neuromuscular junction model based on primary human muscle cells and developed a novel muscle cell-based assay (CBA) for the detection of neuromuscular autoantibodies. How might this impact on clinical practice or future developments?The CBA detected autoantibodies in patients with AChR- or MuSK-positive MG with high sensitivity and specificity and demonstrated their functional pathogenic effects on AChR clustering. This antigen-independent approach may improve the detection and functional characterization of MG autoantibodies, particularly in patients who are seronegative in current diagnostic assays. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/743478v1_ufig1.gif" ALT="Figure 1000"> View larger version (38K): org.highwire.dtl.DTLVardef@c2e1fforg.highwire.dtl.DTLVardef@8336fforg.highwire.dtl.DTLVardef@8fa2b0org.highwire.dtl.DTLVardef@1d94e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Preclinical efficacy of combinatorial B7-H3 CAR T cells and ONC206 against diffuse intrinsic pontine glioma

BackgroundDiffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor affecting over 300 children annually in the United States. Chimeric antigen receptor (CAR) T cells are a targeted immune effector cell therapy with substantial clinical benefit against hematologic cancers. Against CNS tumors, CAR T cells targeting B7-H3, a protein highly expressed on DIPG, have rapidly advanced from preclinical studies to clinical trials. BrainChild-03 (NCT04185038), a phase 1 trial of repeatedly delivered intracerebroventricular (ICV) B7-H3-targeting CAR T cells (B7-H3 CAR T cells), demonstrated tolerability and potential efficacy for children and young adults with DIPG. However, clinical benefits were not uniformly seen, and multi-agent treatment strategies may be required against such an aggressive disease. Here, we combined B7-H3 CAR T cells with ONC206, an imipridone molecule also under clinical investigation. MethodsWe tested B7-H3 CAR T cells combined with ONC206 across multiple DIPG cell cultures and orthotopic xenograft mouse models. ResultsB7-H3 CAR T cell monotherapy induced robust cytotoxicity while ONC206 treatment resulted in significant mitochondrial dysfunction against DMG/DIPG cells. The combination of low effector-to-target ratios of B7-H3 CAR T cells and IC50 concentrations of ONC206 led to significantly enhanced cytotoxicity in vitro (p<0.003) and increased IL-2, IL-29, VEGF-A, and Granzyme B levels. In vivo combinatorial studies of ONC206 and a single ICV dose of B7-H3 CAR T cells significantly extended survival in multiple DIPG xenograft mouse models (p<0.05). ConclusionsB7-H3 CAR T cells combined with ONC206 is a feasible and efficacious multi-agent approach against multiple DIPG models. Importance of the studyDiffuse intrinsic pontine glioma (DIPG) is a fatal pediatric brain tumor. While B7-H3 CAR T cells have shown tolerability and potential benefit in early trials, combinatorial regimens may be required for consistent cures against this aggressive disease. This study demonstrates that a preclinical therapeutic regimen of B7-H3 CAR T cells with ONC206, a second-generation imipridone, increases anti-tumor efficacy in vitro and in orthotopic DIPG mouse models. To our knowledge, this is the first study to evaluate ONC206 in combination with CAR T cells. Our findings provide a preclinical roadmap for evaluating small molecules with CAR T cells to interrogate both their combined benefit and the effect of small molecules on T cells themselves. This work offers a biologically-informed, clinically translatable strategy integrating small molecule therapeutics with CAR T cell therapy and support the development of multi-agent immunotherapy trials for children with DIPG and other high-grade brain and spinal cord tumors. Key PointsO_LIB7-H3 CAR T cells are cytotoxic against preclinical DMG models. C_LIO_LIONC206 causes metabolic apoptosis in preclinical DMG models. C_LIO_LIB7-H3 CAR T cells and ONC206 have combinatorial efficacy against DMG. C_LI

cancer biology↗

bioRxiv2025

Plectin affects cell viscoelasticity at small and large deformations

Plectin is a giant protein of the plakin family that crosslinks the cytoskeleton of mammalian cells. It is expressed in virtually all tissues and its dysfunction is associated with various diseases such as skin blistering. There is evidence that plectin regulates the mechanical integrity of the cytoskeleton in diverse cell and tissue types. However, it is unknown how plectin modulates the mechanical response of cells depending on the frequency and amplitude of mechanical loading. Here we demonstrate the role of plectin in the viscoelastic properties of fibroblasts at small and large deformations by quantitative single-cell compression measurements. To identify the importance of plectin, we compared the mechanical properties of wild type (Plec+/+) fibroblasts and plectin knockout (Plec-/-) fibroblasts. We show that plectin knockout cells are nearly 2-fold softer than wild type cells, but their strain-stiffening behaviour is similar. Plectin deficiency also caused faster viscoelastic stress relaxation at long times. Fluorescence recovery after photobleaching experiments indicated that this was due to 3-fold faster actin turnover. Short-time poroelastic relaxation was also faster in Plec-/- cells as compared to Plec+/+ cells, suggesting a more sparse cytoskeletal network. Confocal imaging indicated that this was due to a marked change in the architecture of the vimentin network, from a fine meshwork in wild type cells to a bundled network in the plectin knockout cells. Our findings therefore indicate that plectin is an important regulator of the organization and viscoelastic properties of the cytoskeleton in fibroblasts. Our findings emphasize that mechanical integration of the different cytoskeletal networks present in cells is important for regulating the versatile mechanical properties of cells. SIGNIFICANCEMammalian cells combine superior mechanical strength with the ability to actively deform themselves. They owe this paradoxical mechanical behaviour to their cytoskeleton, an intracellular web of protein filaments that includes actin filaments and intermediate filaments. It is known that both cytoskeletal filament types contribute to cell stiffness on their own, but the impact of their mechanical integration via cytoskeletal crosslinker proteins remains unknown. Here we test the effect of crosslinking of actin and vimentin intermediate filaments by the crosslinker protein plectin in fibroblasts by single-cell compression measurements. By comparing normal cells and cells in which plectin is knocked out, we find that plectin significantly increases cell stiffness and provides a protective mechanism against actin network disruption by compressive loading.

biophysics↗

bioRxiv2025

Impaired autophagic flux in skeletal muscle of plectin-related epidermolysis bullosa simplex with muscular dystrophy

BackgroundPlectin, a multi-functional cytolinker and intermediate filament (IF) stabilizing protein, is essential for muscle fiber integrity and function. Mutations in the human plectin gene (PLEC) cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). The disorganization and aggregation of desmin IFs in conjunction with degenerative changes of the myofibrillar apparatus are key features in the skeletal muscle pathology of EBS-MD. We performed a comprehensive analysis addressing protein homeostasis in this rare protein aggregation disease by using human EBS-MD tissue, plectin knock-out mice, and plectin-deficient cells. MethodsProtein degradation pathways were analyzed in muscles from EBS-MD patients, muscle-specific conditional plectin knockout (MCK-Cre/cKO) mice, as well as immortalized plectin-deficient (Plec-/-) myoblasts by electron and immunofluorescence microscopy. To obtain a comprehensive picture of autophagic processes, we evaluated the transcriptional regulation and expression levels of autophagic markers in plectin-deficient muscles and myoblasts (RNA-Seq, qRT-PCR, immunoblotting). Autophagic turnover was dynamically assessed by measuring baseline autophagy as well as specific inhibition and activation in mCherry-EGFP-LC3B-expressing Plec+/+and Plec-/- myoblasts, and by monitoring primary wild-type (WT) and plectin-deficient (P0) myoblasts using organelle-specific dyes. Analyses of chloroquine (CQ)-treated MCK-Cre/cKO mice corroborated that loss of plectin coincides with impaired autophagic clearance. ResultsOur study identified massive accumulation of degradative vacuoles as well as LC3 and SQSTM1-positive patches in EBS-MD patient and MCK-Cre/cKO mouse muscles and Plec-/- myoblasts. While the transcriptional regulation of autophagy-related proteins remained largely unaltered, protein levels of downstream targets of the autophagosomal degradation machinery were elevated in MCK-Cre/cKO muscle lysates (e.g. LAMP2, BAG3, and SQSTM1 to [~]160, [~]150, and [~]140% of control samples, respectively; P<0.05). Autophagosome turnover was compromised in mCherry-EGFP-LC3B-expressing Plec-/-myoblasts compared to Plec+/+ cells ([~]40% reduction in median red:green ratio, reduced puncta number, smaller puncta; P<0.01). By labelling autophagic compartments with CYTO-ID dye or lysosomes with LYSO-ID, we found reduced signal intensities in P0 cells (P<0.001). Treatment of primary myoblasts with CQ led to drastic swelling of autophagic vacuoles in WT myoblasts, while the swelling in P0 cells was moderate, establishing a defect in their autophagic clearance. Finally, CQ-treatment of MCK-Cre/cKO mice reassured in vivo the concept that autophagic flux is impaired in plectin-deficient muscles. ConclusionsOur work demonstrates that the characteristic protein aggregation pathology in EBS-MD is linked to an impaired autophagic flux. The obtained results open a new perspective on the understanding of the protein aggregation pathology in plectin-related disorders and provides a basis for further pharmacological intervention.

cell biology↗

bioRxiv2024

Fine Tuning Genetic Circuits via Host Context and RBS Modulation

The choice of organism to host a genetic circuit - the chassis - is often defaulted to model organisms due to their amenability. The chassis-design space has therefore remained underexplored as an engineering variable. In this work, we explored the design space of a genetic toggle switch through variations in nine ribosome binding sites compositions and three host contexts, creating 27 circuit variants. Characterization of performance metrics in terms of toggle switch output and host growth dynamics unveils a spectrum of performance profiles from our circuit library. We find that changes in host-context causes large shifts in overall performance, while modulating ribosome binding sites leads to more incremental changes. We find that a combined ribosome binding site and host-context modulation approach can be used to fine tune the properties of a toggle switch according to user-defined specifications, such as towards greater signaling strength, inducer sensitivity or both. Other auxiliary properties, such as inducer tolerance, are also exclusively accessed through changes in host-context. We demonstrate here that exploration of the chassis-design space can offer significant value, reconceptualizing the chassis-organism as an important part in the synthetic biologists toolbox with important implications for the field of synthetic biology. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/604438v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1ff45d0org.highwire.dtl.DTLVardef@4060d7org.highwire.dtl.DTLVardef@95d5d8org.highwire.dtl.DTLVardef@10a98d5_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2023

YidC from Escherichia coli forms an ion-conducting pore upon activation by ribosomes.

The universally conserved protein YidC aids the insertion and folding of transmembrane polypeptides independently or as a part of the SecYEG translocon complex. In the former scenario, the lipid-exposed YidC surface equipped with a highly conserved positively charged arginine is thought to facilitate membrane insertion of the nascent chain by providing a countercharge for the acidic residues at the polypeptides N-terminal region. Here we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon binding a ribosome or ribosome-nascent chain complex (RNC). This pore is closed in the absence of ribosomes. As this pore is not visible in the published monomeric YidC structure, we used AlphaFold to construct the model of a parallel YidC dimer. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, and single-molecule fluorescence microscopy. In the dimeric model, the conserved positively charged arginine and many residues interacting with nascent chains point into the putative pore. This result suggests the possibility of an alternative mode of YidC-assisted membrane protein insertion.

biophysics↗

bioRxiv2020

The non-typeable Haemophilus influenzae major adhesin Hia is a dual function lectin that binds to human-specific respiratory tract sialic acid glycan receptors.

NTHi is a human-adapted pathogen that colonises the human respiratory tract. Strains of NTHi express multiple adhesins, however there is a unique, mutually exclusive relationship between the major adhesins Hia and HMW1/2. Approximately 25% of NTHi strains express Hia, a phase-variable autotransporter protein, and which has a critical role in colonisation of the host nasopharynx. The remaining 75% of strains express HMW1/2. Previous work has shown that the HMW1 and HMW2 proteins mediate binding to 2,3- and 2,6-linked sialic acid glycans found in the human respiratory tract. Here we show that that the high affinity binding domain of Hia, binding domain 1 (BD1) is responsible for binding to 2,6-sialyllactosamine glycans. BD1 is highly specific for glycans that incorporate the form of sialic acid expressed by humans, N-acetylneuraminic acid (Neu5Ac). We further show that Hia has lower affinity binding activity for 2,3-linked sialic acid and that this binding activity is mediated via a distinct domain. Thus, Hia with its dual binding activities functionally mimics the combined activities of the HMW1 and 2 adhesins. In addition, we show that Hia has a role in biofilm formation by strains of NTHi that express the adhesin. Knowledge of the binding affinity of a major NTHi adhesin, and putative vaccine candidate, will direct and inform development of future vaccines and therapeutic strategies for this important pathogen. ImportanceHost-adapted bacterial pathogens like NTHi have evolved specific mechanisms to colonize their restricted host niche. Relatively few of the adhesins expressed by NTHi have been characterized as regards their binding affinity at the molecular level. In this work we show that the major NTHi adhesin, Hia, preferentially binds to Neu5Ac-2,6-sialyllactosamine, the form of sialic acid expressed in humans. The receptors targeted by Hia in the human airway mirror those targeted by influenza A virus and indicates the broad importance of sialic acid glycans as receptors for airway pathogens.

microbiology↗