Biology subjects

Metz, J.

Publications and source records attributed to Metz, J..

17 recordsLinked to original sources

bioRxiv2026

IL-27 Reduces Cell-Surface LDLR in Human Macrophages and Restricts VSV-G-Pseudotyped HIV infection

The low-density lipoprotein receptor (LDLR) is a major cellular receptor for vesicular stomatitis virus glycoprotein (VSV-G), which is widely used to pseudotyped virus vectors. A replication incompetent VSV-G-pseudotyped HIV (VSV-G-HIVLuc) has been used in HIV infection studies. Although IL-27-polarized macrophages induce multiple antiviral genes and resist replication-competent HIV infection, their impact on VSV-GHIVLuc infection has not been studied. Here, we evaluated the role of IL-27 polarization on VSV-G-HIVLuc infection. Infection was monitored by luciferase activity. Cell-surface LDLR was analyzed by flow cytometry. LDLR transcripts and protein expression were quantified by qRT-PCR and Western blotting, respectively. Viral binding was assessed by measuring cell-associated p24 antigen, while post-entry events were evaluated by quantifying late reverse-transcription products. VSV-G-HIVLuc infection in IL-27-polarized M-CSF-induced macrophages (27M-Mac) and IL-27-polarized GM-CSFinduced macrophages (27GM-Mac) was reduced to 5.8{+/-}0.4% and 5.9{+/-}2.1%, respectively (both p<0.0001), compared to unpolarized macrophages. 27M-Mac and 27GM-Mac reduced cell-surface LDLR to 38.3{+/-}2.6% (p<0.01) and 31.4{+/-}3.2% (p<0.01). Western blotting showed that total LDLR protein decreased to 48{+/-}0.8% in 27M-Mac (p<0.01), whereas 27GM-Mac showed no significant change. However, LDLR transcripts increased approximately 1.6-1.9-fold following IL-27 treatment in both macrophage types (p<0.01). Viral binding to 27M-Mac decreased modestly, while binding to 27GM-Mac decreased significantly to 67.5 {+/-} 7.5% of that to GM-Mac (p<0.05, n=3). In contrast, late reverse-transcription products decreased to 23{+/-}2.1% (p<0.0001) and 44{+/-}1.5% (p<0.0001). These findings demonstrate that IL-27 downregulates cell-surface LDLR expression in macrophages despite increasing LDLR transcript abundance, suggesting that IL-27 restricts VSV-G-pseudotyped Lentiviral infection through both receptor-associated and additional post-entry mechanisms.

immunology↗

bioRxiv2026

Proton FLASH radiotherapy enhances control of triple-negative breast cancer through STING-IRF3 and CD8+ T-cell immunity

FLASH radiotherapy delivers radiation at ultra-high dose rates and has been demonstrated to spare normal tissue compared to standard radiotherapy, but it is not known if dose rate also modifies tumor response. Here we compare a single 13.5 Gy fraction of proton irradiation delivered at FLASH (F-PRT) or Standard (S-PRT) dose rate in immunocompetent C57BL/6 mice bearing EO771 or AT3 triple-negative mammary tumors. At this identical physical dose, F-PRT delays tumor growth more than S-PRT at both heterotopic and orthotopic sites. The effect is largest in EO771, where F-PRT also prolongs tumor-volume endpoint-free survival and reduces the emergence of lung metastases relative to S-PRT. F-PRT induces earlier intratumoral STING expression and IRF3 nuclear translocation, higher type I interferon levels and greater CD8+ T-cell infiltration. CD8+ T-cell depletion or systemic STING inhibition abolishes the F-PRT advantage. Combined with anti-PD-1 and agonistic anti-CD40, both modalities produce durable complete responses that reject contralateral rechallenge, but F-PRT limits tumor progression before response and accelerates regression. FLASH proton radiotherapy not only improves normal-tissue tolerance, but also antitumor immunity, suggesting that ultra-high dose rate could widen the therapeutic window from both sides.

cancer biology↗

bioRxiv2026

Fractionated proton and photon FLASH irradiation mitigates radiation-induced lymphopenia through kinetic sparing of circulating lymphocytes

Background and purpose: Radiation-induced lymphopenia is associated with adverse outcomes in thoracic malignancies. FLASH radiotherapy delivers radiation over a timescale of hundreds of milliseconds, potentially reducing the fraction of irradiated circulating lymphocytes. In this study, we investigated whether FLASH mitigates lymphopenia after thoracic irradiation delivered with protons or photons. Materials and methods: C57BL/6 mice received three 13.5-Gy whole-heart fractions at 48-hour intervals using FLASH or standard dose-rate proton irradiation at the University of Pennsylvania (n=15), with photon validation at Queen's University Belfast (n=72). Leukocytes and CD4 T cells, CD8 T cells, B cells, and NK cells were quantified by hemocytometer and flow cytometry. A continuous-time Markov model simulated lymphocyte trafficking, dose accumulation, and post-irradiation recovery. Results: FLASH attenuated leukocyte depletion across both proton and photon irradiation modalities. In the proton cohort, white blood cell counts were significantly higher after FLASH at D1, D3, D7, and D14; CD4 T cells and NK cells were preserved through D14, while CD8 T cell sparing persisted through D21. Photon FLASH preserved CD45 leukocytes at D1, D3, D7, and D21, with sustained CD8 sparing at D21. Modeling showed that FLASH shifted the lymphocyte dose distribution toward lower exposures, increasing the proportion of lymphocytes receiving <1 Gy from 2.4% to 16.4%, and reduced the proportion of lymphocytes repeatedly irradiated across all three fractions from 36.3% at standard dose rate to 9.18%, despite similar median cumulative doses. The spleen contributed substantially to cumulative lymphocyte dose, and marrow-entering lymphocytes displayed a more high-dose-enriched distribution after FLASH irradiation. Conclusion: FLASH consistently mitigated radiation-induced lymphopenia for proton and photon modalities, with durable CD8 T cell preservation. These findings support a kinetic mechanism and provide a rationale for combining FLASH radiotherapy with immune-sparing planning and immunotherapy.

cell biology↗

bioRxiv2026

The Onset of Myeloid Differentiation in Hematopoietic Progenitors In Vivo is Coordinated with S-phase Progression

Hematopoiesis produces diverse cell types in large quantities. How hematopoietic stem cells (HSCs) initiate lineage differentiation in vivo is not known. Here, we jointly measure chromatin accessibility and transcriptomes in single stem and progenitor cells and uncover a tight interplay between cell-cycle progression and the onset of differentiation. The cell cycle continuously accelerates as HSCs transition to uncommitted multipotent progenitors. In these uncommitted progenitors, proliferative activation is followed by the opening of myeloid-specific chromatin loci in S phase and, subsequently, myeloid gene expression. S phase-coupled onset of myeloid differentiation becomes quantitatively enhanced in emergency myelopoiesis. At the molecular level, we find that chromatin occupancy by HOXA9 - a key transcription factor of uncommitted progenitors - predicts whether the transition through S phase will initiate myelopoiesis, or whether the uncommitted progenitor state will be retained. Taken together, our findings provide a mechanistic framework for the onset of myeloid lineage differentiation in vivo.

systems biology↗

bioRxiv2026

Mesenchymal stroma cell-derived stem cell factor mediates cross-species compatibility of the hematopoietic stem cell niche.

The regulation of human hematopoietic stem cell (HSC) function within its native bone marrow microenvironment remains poorly understood due to the limitations of existing humanized models. Here, we present a non-conditioned human HSC xenotransplantation platform that serves as a physiologically relevant in vivo surrogate to study these complex cellular interactions. Using this system, we uncover a dynamic, cross-species communication between human HSCs and the murine niche, revealing a profound cellular and molecular plasticity of the bone marrow microenvironment in response to humanization. Upon engraftment, platelet-derived growth factor receptor alpha positive (Pdgfra+) mesenchymal stromal cells (MSCs) undergo significant numerical expansion and a major transcriptional shift, transitioning from a mixed adipo- and osteo-primed state toward a leptin receptor positive (Lepr+) predominantly adipo-primed, hematopoiesis-supporting signature. Functional validation demonstrates that this niche plasticity is a critical determinant of stem cell engraftment: the targeted depletion of Lepr+ MSCs or the genetic deletion of stem cell factor results in the rapid mobilization and lack of engraftment of human HSCs, respectively. Our findings establish niche plasticity as a primary regulator of human HSC function and demonstrate the utility of this cross-species platform as a modular genetic toolbox for precise in vivo engineering of the bone marrow microenvironment. Ultimately, this adaptable system provides a powerful framework for elucidating human stem cell biology and advancing bone marrow transplantation therapies.

cell biology↗

bioRxiv2026

Uncompromised, multimodal, multiscale structural analysis of the hierarchically organization in mineralized tissues

We present a live-to-cryo correlative imaging workflow for multiscale structural and chemical analysis of biological tissues in their near-native state. The method integrates live super-resolution fluorescence microscopy, live and cryogenic Raman spectroscopy, and targeted cryogenic focused ion beam/scanning electron microscopy, transmission electron microscopy, electron tomography, energy dispersive X-ray spectroscopy, and electron diffraction. This approach enables precise 3D targeting and nanoscale imaging of selected regions across four orders of magnitude in spatial resolution, while preserving ultrastructure and chemical composition. Using regenerating zebrafish scales as a benchmark, we visualize collagen fibril orientation, local matrix density, and mineral composition within the extracellular matrix. We identify a plywood-like architecture of unmineralized collagen with orientation-independent density variation, and reveal curved, acidic phosphate-rich mineral platelets aligned with collagen fibrils. This workflow establishes a generalizable strategy for comprehensive 3D correlative analysis of hybrid tissues, and opens new opportunities for studying native structure-function relationships at the interface of biology and materials science.

biophysics↗

bioRxiv2025

Membrane curvature regulates Ups1 dependent phosphatidic acid transfer across lipid bilayers

Mitochondria are essential organelles in eukaryotic cells, enclosed by two membranes with distinct compositions and functions. In addition to the endoplasmic reticulum, mitochondria are major sites of cellular lipid production. Cardiolipin, for example, is exclusively synthesized in the mitochondrial inner membrane. This requires the precursor lipid phosphatidic acid to be imported from the endoplasmic reticulum to the mitochondrial outer membrane. Subsequently phosphatidic acid is transferred to the inner membrane by the lipid transfer protein Ups1/PRELID1. The regulation of this process, the role of membrane physico-chemical properties, and the mechanisms by which energy barriers are overcome during lipid extraction and insertion remain poorly understood. Here, we demonstrate that Ups1 exhibits a strong preference for binding to positively curved membrane regions. Our findings reveal that phosphatidic acid extraction is energetically favored at these membrane domains, leading to enhanced lipid transfer between membranes with high positive curvature and we show that events at the donor membrane are rate limiting for the transfer cycle. Our data suggest that Ups1 membrane binding is modulated by pH, lipid composition, and membrane morphology, pointing to a complex, multipartite regulatory network underlying intra-mitochondrial lipid transfer.

biochemistry↗

bioRxiv2024

Molecular insight into the network of Drosophila cytoplasmic piRNA pathway proteins through a combination of systematic interaction screening and structural prediction

piRNA-bound PIWI proteins mediate the silencing of transposons at both the transcriptional and post-transcriptional levels, processes that are critical for genome integrity and fertility in animals. While numerous additional proteins are known to be essential for piRNA biogenesis and function in Drosophila and other animals, their molecular and mechanistic functions have remained largely unknown. To improve our molecular understanding of the Drosophila piRNA pathway, we used a cell culture-based protein-protein interaction assay called ReLo to perform a systematic pairwise interaction screen involving 22 factors operating in the cytoplasm, including PIWI proteins, Tudor domain-containing proteins (TDRDs), RNA helicases, and mitochondrial surface proteins. Through additional ReLo interaction testing and structural modeling using AlphaFold-Multimer, we have characterized six protein complexes at the molecular and structural levels. We believe that the results of this screen and our methodological approach are likely to guide future research into the molecular mechanisms underlying piRNA biogenesis and function.

biochemistry↗

bioRxiv2023

Structural basis for binding of Smaug to the GPCR Smoothened and to the germline inducer Oskar

Drosophila Smaug and its orthologs comprise a family of mRNA repressor proteins that exhibit various functions during animal development. Smaug proteins contain a characteristic RNA-binding sterile- motif (SAM) domain and a conserved but uncharacterized N-terminal domain (NTD). Here, we resolved the crystal structure of the NTD of the human SAM domain-containing protein 4A (SAMD4A, a.k.a. Smaug1) to 2.0 [A] resolution, which revealed its composition of a homodimerization D-subdomain and a subdomain with similarity to a PHAT domain. Furthermore, we show that Drosophila Smaug directly interacts with the Drosophila germline inducer Oskar and with the Hedgehog signaling transducer Smoothened through its D-PHAT domain. We determined the crystal structure of the D-PHAT domain of Smaug in complex with a Smoothened -helical peptide to 1.61 [A] resolution. The peptide binds within a groove that is formed by both the D- and PHAT subdomains. Structural modeling supported by experimental data suggested that an -helix within the disordered region of Oskar binds to the D-PHAT domain in a mode similar to Smoothened. Together, our data uncover the N-terminal D-PHAT domain of Smaug as peptide-binding domain.

biochemistry↗

bioRxiv2022

FULLY AUTOMATED POINT SPREAD FUNCTION ANALYSIS USING PYCALIBRATE

Reproducibility is severely limited if instrument performance is assumed rather than measured. Within optical microscopy, instrument performance is typically measured using sub-resolution fluorescent beads. However, the process is performed infrequently as it is requires time and suitably trained staff to acquire and then process the bead images. Analysis software still requires the manual entry of imaging parameters. Human error from repeatedly typing these parameters can significantly affect the outcome of the analysis, rendering the results less reproducibile. To avoid this issue, PyCalibrate has been developed to fully automate the analysis of bead images. PyCalibrate can be accessed either by executing the Python code locally or via a user-friendly web portal to further improve accessibility when moving between locations and machines. PyCalibrate interfaces with the BioFormats library to make it compatible with a wide range of proprietary image formats. In this study, PyCalibrate analysis performance is directly compared with alternative free-access analysis software PSFj, MetroloJ QC and DayBook3 and is demonstrated to have equivalent performance but without the need for user supervision.

bioengineering↗

bioRxiv2022

Precise targeting for 3D cryo-correlative light and electron microscopy volume imaging of tissues using a FinderTOP

Cryo-correlative light and electron microscopy (cryoCLEM) is a powerful strategy to high resolution imaging in the unperturbed hydrated state. In this approach fluorescence microscopy aids localizing the area of interest, and cryogenic focused ion beam/scanning electron microscopy (cryoFIB/SEM) allows preparation of thin cryo-lamellae for cryoET. However, the current method cannot be accurately applied on bulky (3D) samples such as tissues and organoids. 3D cryo-correlative imaging of large volumes is needed to close the resolution gap between cryo-light microscopy and cryoET, placing sub-nanometer observations in a larger biological context. Currently technological hurdles render 3D cryoCLEM an unexplored approach. Here we demonstrate a cryoCLEM workflow for tissues, correlating cryo-Airyscan confocal microscopy with 3D cryoFIB/SEM volume imaging. Accurate correlation is achieved by imprinting a FinderTOP pattern in the sample surface during high pressure freezing, and allows precise targeting for cryoFIB/SEM volume imaging.

cell biology↗

bioRxiv2022

Wheat cells show positional responses to invasive Zymoseptoria tritici

The stomatal complex of grasses consists of two guard cells and two adjacent subsidiary cells that cooperate during stomatal closure. Zymoseptoria tritici, the main causal agent of Septoria tritici blotch in wheat, enters the host via stomata. Here we test the hypothesis that the stomatal complex shows focused sub-cellular responses to invading Z. tritici hyphae. We have combined live-cell transmission light microscopy, immunofluorescence and CRS microscopy to identify cell wall modifications triggered by hyphal invasion. Furthermore, we have used confocal fluorescence microscopy and automated quantitative image analysis to assess whether host cells respond to hyphae through spatial redistribution of organelles. We find that subsidiary cells construct papillae that are accurately aligned with hyphal position even when hyphae are occluded by guard cells. These are distinct from those induced by powdery mildew, with callose restricted to a crust that surrounds content with a high-amplitude Raman signal in the CH-band. Peroxisome populations in subsidiary cells show distributions with modes weakly correlated with hyphal position but do not differ significantly between compatible and incompatible interactions. Our data suggest local changes to cell wall architecture and focal accumulation of organelles in subsidiary cells could play roles in crop defence during host leaf penetration by Z. tritici. Molecular strategies to amplify these responses may provide novel routes for crop protection.

plant biology↗

bioRxiv2022

RNA binding proteins Smaug and Cup induce CCR4-NOT-dependent deadenylation of the nanos mRNA in a reconstituted system

Posttranscriptional regulation of the maternal nanos mRNA is essential for the development of the anterior - posterior axis of the Drosophila embryo. The nanos RNA is regulated by the protein Smaug. Binding to Smaug recognition elements (SREs) in the nanos 3-UTR, Smaug nucleates the assembly of a larger repressor complex including the eIF4E-T paralog Cup and five additional proteins. The Smaug-dependent complex represses translation of nanos and induces its deadenylation by the CCR4-NOT deadenylase. Here we report an in vitro reconstitution of the Drosophila CCR4-NOT complex and Smaug-dependent deadenylation. We find that Smaug by itself is sufficient to cause deadenylation by the Drosophila or human CCR4-NOT complexes in an SRE-dependent manner. CCR4-NOT subunits NOT10 and NOT11 are dispensable, but the NOT module, consisting of NOT2, NOT3 and the C-terminal part of NOT1, is required. Smaug interacts with the C-terminal domain of NOT3. Both catalytic subunits of CCR4-NOT contribute to Smaug-dependent deadenylation. Whereas the CCR4-NOT complex itself acts distributively, Smaug induces a processive behavior. The cytoplasmic poly(A) binding protein (PABPC) has but a minor effect on Smaug-dependent deadenylation. Among the additional constituents of the Smaug-dependent repressor complex, Cup also facilitates CCR4-NOT-dependent deadenylation, both independently and in cooperation with Smaug.

biochemistry↗

bioRxiv2022

ReLo: a simple colocalization assay to identify and characterize physical protein-protein interactions

The characterization of protein-protein interactions (PPIs) is fundamental for understanding biochemical processes. Many methods have been established to identify and study direct PPIs; however, the screening and investigation of PPIs involving large or poorly soluble proteins remain challenging. As a result, we developed ReLo, a simple, rapid, and versatile cell culture-based method for detecting and investigating interactions in a cellular context. Importantly, our data strongly suggest that with ReLo specifically direct binary PPIs are detected. By applying additional bridging experiments ReLo can also be used to determine the binding topology of subunits within multiprotein complexes. Moreover, ReLo has the potential to identify protein domains that mediate complex formation, screen for interfering point mutations, study interactions that depend on conformation or protein arginine methylation, and it is sensitive to drugs that mediate or interfere with an interaction. Taken together, ReLo is a simple and quick alternative for the study of PPIs particularly when established methods fail.

cell biology↗

bioRxiv2021

Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death

Polyglutamine (polyQ) diseases are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases in humans. Computer-aided drug discovery was implemented to identify human TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined, and Biacore experiments with the RRM were performed. The identified molecules were validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Our work provides a first step towards pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.

molecular biology↗

bioRxiv2020

A Quantitative Measurement to Describe the Relative Proximity of Fluorescent Biomarkers

Determining the relationship between biomarkers via fluorescence microscopy is a key step in the characterisation of cellular phenotypes. We define a simple distance-based measurement termed a perimeter distance mean (PDmean) which quantifies the relative proximity of objects in one fluorescent channel to objects in a second fluorescent channel in 2D or 3D microscopy datasets. PDmean measurements were able to accurately identify known changes in colocalisation in computer-generated and real-world microscopy datasets. We argue that this approach provides substantial advantages over currently used distance-based colocalisation analysis methods. We also introduce PyBioProx, an extensible open-source Python module and graphical user interface that produces PDmean measurements.

bioinformatics↗

bioRxiv2019

Label-free single-cell microfluidics reveals antibiotic transport kinetics in Gram-negative bacteria

The double-membrane cell envelope of Gram-negative bacteria is a formidable barrier to intracellular antibiotic accumulation. A quantitative understanding of antibiotic transport in these cells is crucial for drug development, but this has proved elusive due to the complexity of the problem and a dearth of suitable investigative techniques. Here we combine microfluidics and time-lapse auto-fluorescence microscopy to quantify antibiotic uptake label-free in hundreds of individual Escherichia coli cells. By manipulating the microenvironment, we showed that drug (ofloxacin) accumulation is higher in growing versus non-growing cells. Using genetic knockouts, we provide the first direct evidence that growth phase is more important for drug accumulation than the presence or absence of individual transport pathways. We use our experimental results to inform a mathematical model that predicts drug accumulation kinetics in subcellular compartments. These novel experimental and theoretical results pave the way for the rational design of new Gram-negative antibiotics.

biophysics↗