Biology subjects

Freitag, A.

Publications and source records attributed to Freitag, A..

8 recordsLinked to original sources

bioRxiv2026

The Glucose Transporter GLUT3 Controls Regulatory T Cell Function

Regulatory T (Treg) cells are central mediators of immune tolerance and are generally considered to rely predominantly on mitochondrial metabolism rather than glucose-driven glycolysis. To define the role of glucose metabolism in Treg cells, we investigated the contribution of the hexose transporters GLUT1 and GLUT3. Genetic ablation of GLUT1 in T cells or selectively in Treg cells had minimal impact on Treg cell numbers, phenotype or immune homeostasis, indicating that GLUT1 is largely dispensable in this lineage. By contrast, deletion of GLUT3 in T cells resulted in a marked reduction in Treg cell numbers. However, it remained unclear whether this reduction reflected diminished IL-2 production by GLUT3-deficient conventional T cells or a cell-intrinsic requirement for GLUT3 in Treg cells. To investigate this, we generated mice with Treg cell-specific deletion of GLUT3. These animals developed severe systemic inflammation accompanied by lethal cellular and humoral autoimmunity. Mechanistically, GLUT3-deficient Treg cells exhibited reduced glycolytic activity and mitochondrial respiration, leading to impaired suppressive function and defective effector and follicular Treg cell differentiation. Collectively, our findings demonstrate a non-redundant requirement for GLUT3 in Treg cell metabolic fitness and immune regulation, refining the prevailing view that Treg cells operate largely independently of glucose metabolism. Our data further suggest that therapeutic strategies targeting glucose uptake and glycolysis in autoimmune and inflammatory diseases should account for potential adverse effects on Treg cell-mediated immune tolerance.

immunology↗

bioRxiv2025

Glucose Metabolism Controls Oxidative Burst and Lipid Mediator Production in Neutrophils upon Microbial Challenge

Neutrophils are frontline responders against bacterial and fungal pathogens, requiring rapid energy and biosynthetic precursors to mount effective antimicrobial responses. To meet these demands, they primarily rely on aerobic glycolysis, making glucose uptake essential. Murine and human neutrophils express the glucose transporters GLUT1 and GLUT3; however, their specific roles in neutrophil immunobiology have not yet been fully elucidated. Here, we show that neutrophilic immune responses to Candida albicans and Staphylococcus aureus critically depend on GLUT1/3-dependent glucose uptake and glycolysis. Combined deletion of GLUT1 and GLUT3 almost completely abolished glucose uptake and aerobic glycolysis in murine neutrophils, yet did not impair granulopoiesis, indicating that homeostatic neutrophil development is largely independent of extracellular glucose. By contrast, during microbial challenge, loss of GLUT1/3 severely compromised NADPH-dependent ROS production, oxidative burst and cyclooxygenase-derived lipid mediator (LM) biosynthesis, demonstrating that glucose uptake via GLUT1/3 controls inflammatory effector functions of neutrophils. Moreover, genetic and pharmacologic inhibition of GLUT1/3-mediated glucose utilization reprograms neutrophil metabolism and LM biosynthesis toward an immunomodulatory phenotype. These findings identify a conserved nutrient-sensing metabolic checkpoint that governs neutrophil reprogramming and highlight novel opportunities for therapeutic immunomodulation.

immunology↗

bioRxiv2025

An Integrative Approach to Develop and Characterise Antibodies Against the Cancer Associated Antigen Sialyl Lewis A (CA 19-9)

BackgroundSialyl Lewis A (sLeA), or the CA 19-9 marker, is a tetrasaccharide and a tumour-associated carbohydrate antigen (TACA) overexpressed and abnormally secreted as a serum-borne marker in gastrointestinal malignancies. CA 19-9 is the best validated and only FDA-approved serologic marker clinically used to monitor recurrence, progression, and therapy efficiency in pancreatic ductal adenocarcinoma (PDAC) patients. Due to its altered expression on cancer cells, sLeA is also an attractive target for antibody development. Although recent clinical trials have demonstrated insufficient efficacy of the fully human anti-sLeA 5B1 (MVT-5873) format as a stand-alone drug or an adjuvant therapy in PDAC [1], its safety profile and unique expression in additional malignancies keep CA 19-9 an attractive TACA. Hence, we set out to explore the use of synthetic sLeA to develop novel monoclonal antibodies (mAbs) with improved sLeA recognition and better efficacy. MethodsTwo mAbs targeting sLeA were generated through mice immunisation with synthetic sLeA glycoconjugates, synthetic glycan arrays, and hybridoma technology. We then compared the antigen-binding properties of the newly developed mAbs with the widely used mAb 1116-NS-19- 9 via synthetic glycan arrays, immunohistochemistry (IHC), X-ray crystallography, molecular dynamics (MD) simulation, and Saturation Transfer Difference Nuclear Magnetic Resonance (STD NMR) spectroscopy. ResultsThe newly generated mAbs demonstrated improved affinity and specificity for both synthetic and native sLeA, surpassing the performance of the established mAb 1116-NS-19-9. First, synthetic glycan arrays, surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC) assays confirmed superior antigen-binding properties to synthetic sLeA. In particular, the mAb designated GB11 demonstrated markedly enhanced binding to native sLeA ectopically expressed in B16 melanoma cells. To elucidate the structural origin of GB11s improved antigen binding, we conducted high-resolution mapping of the molecular recognition patterns between sLeA and the different antibodies using X-ray crystallography and STD NMR. These analyses revealed subtle yet critical differences in the glycan engagement and identified key structural features underlying GB11s enhanced recognition of sLeA. MD simulations further supported these observations, indicating distinct orientations of sLeA within the binding pockets of each mAb. ConclusionOur results suggest better recognition of the sLeA antigen by the newly generated GB11 antibody and provide a detailed high-resolution elucidation of the molecular interactions behind it. Our study may provide a novel tool with improved theranostic properties against sLeA-overexpressing malignancies.

cancer biology↗

bioRxiv2024

Do morphologically distinct groups correspond to reproductively isolated species? A case study in Myrmica ants from Switzerland

The most widely used definition of a species is that it is reproductively isolated from other populations. Yet, most species are described on the basis of morphological criteria, and reproductive isolation is seldom tested. Using the ant genus Myrmica Latreille (Hymenoptera, Formicidae) as a model, we ask whether species described as distinct based on (often subtle) morphological differences indeed form reproductively isolated lineages. We collected and morphologically identified 918 Myrmica ants from a 3212 km2 area in Switzerland. We then combined DNA barcoding (based on COI) and RAD sequencing to identify genetically isolated lineages. Out of the 14 morphological species identified, 13 formed genetically differentiated lineages, while the last one was not supported by our genetic data. Overall, the morphological identification was congruent with genetic lineage delineation for 94.9% of individuals. Our dataset also allowed us to screen for cryptic lineages in the five most frequent species, including in M. scabrinodis where cryptic lineages were previously suggested, but we found no evidence for cryptic species. Overall, our results indicate that morphology parallels genetic isolation in the studied species. However, an integrative approach combining morphological identification with nuclear marker genotyping is necessary for confident species identification of all individuals. Finally, our results provide a library of validated COI barcodes for future Myrmica specimen identification.

evolutionary biology↗

bioRxiv2024

LEVERAGING CITIZEN SCIENCE TO ASSESS RICHNESS, DIVERSITY, AND ABUNDANCE IN ANT COMMUNITIES

AO_SCPLOWBSTRACTC_SCPLOWCitizen science is a key resource in overcoming the logistical challenges of monitoring biodiversity. While datasets collected by groups of volunteers typically have biases, recent methodological and technological advances provide approaches for accounting for such biases, particularly in the context of modelling species distributions and diversity. Specifically, data integration techniques allow for the combination of scientifically collected datasets with haphazardly sampled presence-only datasets created by most citizen science initiatives. Here, we use a hierarchical Bayesian framework to integrate a set of ant presences collected by citizen scientists in the Vaud canton (Switzerland) with ant colony density data collected concurrently in the same region following a scientific sampling design. The community-level Poisson point process model included species-specific responses to the local (1.2 m2) and regional (1 km2) environment, with the presence-only samples incorporated at the regional scale to predict local and regional ant communities. At the regional scale, species richness followed a hump-shaped pattern and peaked near 1000 m while abundance increased with elevation. Low elevation and montane ant communities were composed of distinct species assemblages. At the local scale, the link between elevation and richness, diversity, and abundance was weak. At low elevations, local plots varied both in total abundance and species composition, while at higher elevations, the species composition was less variable. The citizen science dataset showed a general tendency toward under-representation of certain species, and heavy spatial sampling bias. Nonetheless, the inclusion of the citizen science data improved predictions of local communities, and also reduced susceptibility to over-fitting. Additionally, the citizen science dataset included many rare species not detected in the structured abundance dataset. The model described here illustrates a framework for capitalizing on the efforts of citizen scientists to better understand the patterns and distribution of biodiversity.

ecology↗

bioRxiv2022

Structure and function of Plasmodium actin II in the parasite mosquito stages

Actins are filament-forming, highly-conserved proteins in eukaryotes. They are involved in essential processes in the cytoplasm and have also nuclear functions. Malaria parasites (Plasmodium spp.) have two actin isoforms that differ from each other and from canonical actins in structure and filament-forming properties. Actin I has an essential role in motility and is fairly well characterized. The structure and function of actin II are not as well understood, but mutational analyses have revealed two essential functions in male gametogenesis and in the zygote. Here, we present expression analysis, high-resolution filament structures, and biochemical characterization of Plasmodium actin II. We show that it is expressed in male gametocytes and zygotes and associated with the nucleus in both stages in filament-like structures. Unlike actin I, actin II readily forms long filaments in vitro, and near-atomic structures in the presence or absence of jasplakinolide reveal very similar structures. Small but significant differences compared to other actins in the openness and twist, the active site, the D-loop, and the plug region contribute to filament stability. Mutational analyses suggest that long and stable filaments are necessary for male gametogenesis, while the second function in the zygote stage also requires finetuned regulation by methylation of histidine 73. Actin II polymerizes via the classical nucleation-elongation mechanism and has a critical concentration of ~0.1 M at the steady-state, like actin I and canonical actins. Similarly to actin I, dimers are a stable form of actin II at equilibrium. Significance statementMalaria is a parasitic infection caused by Plasmodium spp., which belong to the phylum Apicomplexa. In 2020, approximately 627000 people died from nearly 241 million malaria cases registered worldwide. In contrast to other apicomplexan parasites, Plasmodium spp. encode two actin isoforms. While actin I is part of the glideosome complex, essential for locomotion, actin II is present in the mosquito stages, where it has functions in gametogenesis and in the zygote. The exact function of actin II is still unclear, and information at the molecular level is limited. We show here that actin II is associated with the nucleus in gametocytes and zygotes and performs specific functions requiring both long filaments and fine-tuning by methylation of histidine 73. We determined the structures of the filamentous form of actin II at near-atomic resolution and characterized its polymerization properties in vitro. Our study provides a molecular basis for the differences between actins of the malaria parasite and humans, as well as between the two parasite actin isoforms. Furthermore, the in vivo studies provide insights into the function of actin II in the parasite.

biochemistry↗

bioRxiv2022

Thiol-mediated Uptake of a Cysteine-containing Nanobody for Anti-Cancer Drug Delivery

The identification of tumor-specific biomarkers is one of the bottlenecks in the development of cancer therapies. Previous work revealed altered surface levels of reduced/oxidized cysteines in many cancers due to overexpression of redox-controlling proteins such as protein disulfide isomerases on the cell surface. Alterations in surface thiols can promote cell adhesion and metastasis, making thiols attractive targets for treatment. Only a few tools are available to study surface thiols on cancer cells and exploit them for theranostics. Here, we describe a nanobody (CB2) that recognizes B cell lymphoma in a thiol-dependent manner. CB2 binding strictly requires the presence of a non-conserved cysteine in the antigen-binding region and correlates with elevated surface levels of free thiols on B cell lymphoma compared to healthy lymphocytes. Nanobody CB2 can induce complement-dependent cytotoxicity against lymphoma cells when functionalized with synthetic rhamnose trimers. Lymphoma cells internalize CB2 in a thiol-mediated manner such that the nanobody can be used to deliver cytotoxic agents. Hence, surface thiols can be used as lymphoma biomarkers and targeted by thiol-binding nanobodies. Functionalization of internalizable CB2 is the basis for a range of diagnostic and therapeutic applications of this thiol-binding nanobody. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=69 SRC="FIGDIR/small/497993v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1f47ba9org.highwire.dtl.DTLVardef@1e2bd41org.highwire.dtl.DTLVardef@f7151eorg.highwire.dtl.DTLVardef@18b7681_HPS_FORMAT_FIGEXP M_FIG C_FIG SynopsisNanobody CB2 specifically binds and internalizes into B cell lymphoma via thiol-based interactions. Functionalized CB2 can be used for complement recruitment or drug delivery to lymphoma cells.

cancer biology↗

bioRxiv2018

Identification of Miro as a mitochondrial receptor for myosin XIX

Mitochondrial distribution in cells is critical for cellular function and proper inheritance during cell division. In mammalian cells, mitochondria are transported predominantly along microtubules by kinesin and dynein and along actin filaments by myosin. Myosin XIX (Myo19) associates with the outer mitochondrial membrane, but no specific receptor has been identified. Using proximity BioID labeling, we identified Miro-1 and Miro-2 as potential binding partners of Myo19. Interaction studies show that Miro-1 binds to a C-terminal fragment of the Myo19 tail region and that Miro recruits the Myo19 tail in vivo. This recruitment is regulated by the nucleotide-state of the N-terminal Rho-like GTPase domain of Miro. Notably, Myo19 protein stability in cells depends on its association with Miro. Finally, Myo19 regulates the subcellular distribution of mitochondria. Downregulation, as well as overexpression, of Myo19 induces perinuclear collapse of mitochondria, phenocopying the loss of kinesin KIF5 or its mitochondrial receptor Miro. These results suggest that Miro coordinates microtubule- and actin-based mitochondrial movement.

cell biology↗