Biology subjects

Cordes, T.

Publications and source records attributed to Cordes, T..

At least 19 recordsLinked to original sources

bioRxiv2026

Extracellular citrate modulates glutamine metabolism in human macrophages during infection

Citrate is a central metabolite linking tricarboxylic acid (TCA) cycle activity to energy and lipid metabolism and supports the synthesis of inflammatory mediators, including itaconate, in macrophages. While citrate is primarily generated endogenously, extracellular citrate levels are elevated under pathological conditions such as citrate transporter disorder. Cells import extracellular citrate through SLC13 transporters, including the sodium-dependent citrate transporter NaCT (encoded by SLC13A5). However, whether macrophages take up extracellular citrate and how this affects metabolism and function remains unclear. Here, we combined mass spectrometry and tracing approaches to investigate the metabolic fate of citrate in human macrophage cell lines, primary, and iPSC-derived macrophages. We demonstrate that cells take up extracellular citrate, which was enhanced under metabolic stress conditions. Exogenous citrate was not substantially utilized as a carbon source but selectively altered glutamine metabolism and responses to bacterial infection with Salmonella enterica Typhimurium and Legionella pneumophila Corby. Our work identifies extracellular citrate as a context-dependent regulator in macrophages that decouples uptake from metabolic utilization. HighlightsO_LIMacrophages import extracellular citrate via SLC13 transporters C_LIO_LIExtracellular citrate accumulates under hypoxia and inflammatory activation C_LIO_LIExtracellular citrate does not fuel central carbon metabolism in human macrophages C_LIO_LICitrate modulates glutamine immunometabolism and modulates immune responses C_LI eTOC blurbVo{beta}-Willenbockel et al. demonstrate that human macrophages accumulate extracellular citrate without using it as a major carbon source. Instead, citrate modulates glutamine utilization, inflammatory responses, and host-pathogen interactions revealing a context-dependent regulatory role for extracellular metabolites in immune cell function.

cell biology↗

bioRxiv2025

A systematic approach for the purification of fluorophore-labelled proteins via anion exchange chromatography

Forster resonance energy transfer (FRET), an optical distance ruler, provides the unique ability to monitor molecular interactions and conformational changes in single biomacromolecules and multi-subunit complexes. It has proven to be a powerful tool to study enzymatic reactions, membrane transport, protein folding, but also the properties of nucleic acids, proteins, molecular motors and many other biological systems and processes. A prerequisite for FRET is targeted (covalent) labelling of macromolecules with two distinct fluorophores. Here, we present a strategy for stochastic labeling of protein residues with the required donor and acceptor dyes via anion exchange chromatography. While this technique has been used before for this purpose, we provide a conceptual basis to systematically design a purification protocol for an arbitrary choice of fluorophores. By characterizing the interaction fluorophore-maleimides with the column material, we are able to select (and predict) which pairs of fluorophores allow successful purification of donor-acceptor-labelled protein with yields up to 98%. We demonstrate the capabilities of the method for bulk and single-molecule FRET assays of various bacterial substrate-binding proteins.

biophysics↗

bioRxiv2025

Single-molecule FRET with a minimalistic 3D-printed setup and dyes in the blue-green spectral region

Forster Resonance Energy Transfer (FRET) is a powerful technique for the detection and characterization of biomolecular interactions and conformational changes with sub-nanometer spatial resolution and a temporal resolution down to the timescale of fluorescence. While the technique is widely adopted in structural biology and biophysics, the evolution of single-molecule FRET has led to experimental setups with sophisticated optical layouts, multi-laser excitation schemes and time-resolved detection electronics. We here present an accessible alternative towards single-molecule FRET based on Brick-MIC, a recently introduced 3D-printed micro-spectroscopy platform. The FRET-Brick uses continuous-wave excitation at 488 nm with a minimal set of opto-mechanical components and photomultiplier detectors (PMTs). With this we were able to significantly reduce the setup complexity retaining single-molecule sensitivity with dyes matching the sensitivity of PMTs. To maximize the photon output of Alexa488, ATTO488 (donors), Alexa555, ATTO542 and Cy3B (acceptors), we introduce ferrocene-derivatives as photostabilizers that increase both dye brightness and remove dark-states. We benchmark the performance of the FRET-Brick with fluorophore-labelled oligonucleotide reference structures also in comparison to accessible volume simulations, and by detecting conformational changes in bacterial substrate binding proteins. Our work demonstrates that qualitative and quantitative smFRET measurements are possible with the minimalistic and cost-effective FRET-Brick.

biophysics↗

bioRxiv2025

Itaconate is metabolized to 2-hydroxymethylsuccinate through a CoA-independent degradation pathway in mitochondria

The immunometabolite itaconate modulates cellular metabolism and is converted into structurally similar C5 dicarboxylates that require advanced analytics to decipher their metabolic fate. Here, we employ high-resolution mass spectrometry and tracing approaches and identify 2-hydroxymethylsuccinate (2HMS) as a previously unrecognized C5 dicarboxylate derived from itaconate. 2HMS synthesis occurs during inflammatory responses and upon itaconate treatment, as detected by 13C itaconate tracing. Pathway analysis reveals that methylglutaconyl-CoA hydratase (AUH) drives 2HMS synthesis through a CoA-independent conversion (CIC) pathway. This pathway is distinct from the CoA-dependent conversion (CDC) pathway that generates mesaconate and itaconyl-CoA influencing B12-dependent processes. In vivo inflammation studies reveal that adipose tissue prefers CIC to produce 2HMS and liver favors CDC-mediated mesaconate synthesis, highlighting tissue-specific itaconate degradation routes. This study identifies a new branch of itaconate metabolism, provides an analytical framework to resolve C5 dicarboxylate networks, and links 2HMS to inflammation and mitochondrial metabolism that might be targeted therapeutically.

cell biology↗

bioRxiv2025

Substrate availability and citrate alter TCA cycle metabolism and SLC13A3 in macrophage immune responses

Cell culture media are commonly formulated to enhance cell growth and often lack the physiological nutrient composition found in human blood plasma. The impact of substrate availability on immune cell metabolism and function remains incompletely understood. Here, we demonstrate that changes in culture medium composition affect mitochondrial metabolic pathways, immune responses, and transport in macrophages. Using mass spectrometry and stable isotope tracing, we identify citrate as a mediator linking extracellular substrate availability to intracellular metabolism. We also observe increased IL-6 secretion and elevated expression of plasma membrane transporter NaDC3 (SLC13A3) under physiological carbon source conditions that are reversed when citrate is excluded from the medium. Our findings demonstrate that extracellular substrate composition shapes macrophage immunometabolism and identify citrate as an extracellular signal that modulates immune responses. This work highlights the importance of physiologically relevant nutrient availability in studying and targeting immunometabolic pathways.

cell biology↗

bioRxiv2025

1-Deoxysphingolipids dysregulate membrane properties and cargo trafficking in the early secretory pathway

1-Deoxysphingolipids are non-canonical sphingolipids linked to several diseases, but their cellular effects are poorly understood. Here, we utilize lipid chemical biology approaches to investigate the role of 1-deoxysphingolipid metabolism on the properties and functions of secretory membranes. We first applied organelle-specific bioorthogonal labeling to visualize the subcellular distribution of metabolically tagged 1-deoxysphingolipids in RPE-1 cells, observing that they are retained in the endoplasmic reticulum (ER). We found that 1-deoxysphingolipids can be transported by the non-vesicular transporter CERT in vitro but are retained at ER exit sites (ERES) in cells, suggesting that they do not efficiently sort into vesicular carriers. Cells expressing disease-associated variants of serine palmitoyl-CoA transferase (SPT) accumulated long-chain 1-deoxysphingolipids, which reduced ER membrane fluidity and enlarged ERES. We observed that the rates of membrane protein release from the ER were altered in response to mutant SPT expression, in a manner that was dependent on the cargo affinity for ordered or disordered membranes. We propose that dysregulation of sphingolipid metabolism alters secretory membrane properties, which can then modulate protein trafficking.

cell biology↗

bioRxiv2025

Minimally Invasive DNA-Mediated Photostabilization for Extended Single-Molecule and Super-resolution Imaging

Photobleaching of fluorescence labels poses a major limitation in single-molecule and super-resolution microscopy. Conventional photostabilization methods, such as oxygen removal and addition of high concentrations of photostabilization additives, often require careful fluorophore selection and can disrupt the biological environment. To address these limitations, we developed a modular and minimally invasive photostabilization approach that utilizes DNA-mediated delivery of a photostabilizer directly to the imaging site. Under lower excitation intensities, the DNA-mediated strategy outperformed solution-based approaches, achieving efficient photostabilization at significantly lower additive concentrations. However, at higher excitation intensities, the stability of a single photostabilizer molecule became the limiting factor. To overcome this and reduce the loss of localizations in DNA-PAINT experiments we have also implemented a recovery scheme where the photostabilizer is continuously replenished at the imaging site. We further extended the approach to cell imaging, demonstrating improved localization rate and precision in 3D-DNA PAINT measurements. DNA-mediated photostabilization offers a promising solution for imaging applications where high additive concentrations are prohibited. Its modularity enables adaptation to various imaging schemes and ultimately expands the repertoire of fluorophores suitable for single-molecule and super-resolution imaging.

biophysics↗

bioRxiv2024

Modular Design and Scaffold-Synthesis of Multi-Functional Fluorophores for Targeted Cellular Imaging and Pyroptosis

Modified commercial fluorophores are essential tools for optical imaging and biomedical research. Their synthetic modification to incorporate new functions, however, remains a challenging task. Conventional strategies rely on linear synthesis in which a parent framework is gradually extended. We here designed and synthesized a versatile library of functional fluorophores via a scaffold-based Ugi four-component reaction (U-4CR). The adaptability of the scaffold is achieved through modification of starting materials. This allows to use a small range of starting materials for the creation of fluorogenic probes that can detect reactive-oxygen species and where the localization into subcellular organelles or membranes can be controlled. We present reaction yields ranging from 60% to 90% and discovered that some compounds can even function as imaging and therapeutic agents via Fenton chemistry inducing pyroptosis in living cancer cells. Our study underlines the potential of scaffold-based synthesis for versatile creation of functional fluorophores and their applications.

cell biology↗

bioRxiv2024

Conserved Lysine in transmembrane helix 5 is key for the inner gating of the LAT transporter BasC

L-amino acid transporters (LATs) play a key role in a wide range of physiological processes. Defects in LATs can lead to neurological disorders and aminoacidurias, while the overexpression of these transporters is related to cancer. BasC is a bacterial LAT transporter with an APC fold. In this study, to monitor the cytoplasmic motion of BasC, we developed a smFRET assay that can characterize the conformational states of the intracellular gate in solution at room temperature. Based on combined biochemical and biophysical data and molecular dynamics simulations, we propose a model in which the conserved lysine residue in TM5 supports TM1a to explore both open and closed states within the cytoplasmic gate under apo conditions. This equilibrium can be altered by substrates, mutation of conserved lysine 154 in TM5, or transport-blocking nanobodies. Overall, these findings provide insights into the transport mechanism of BasC and highlight the significance of the lysine residue in TM5 in the cytoplasmic gating of LATs.

biochemistry↗

bioRxiv2024

Metabolic Deficiencies Underlie Plasmacytoid Dendritic Cell Exhaustion After Viral Infection

Type I Interferons (IFN-I) are central to host protection against viral infections1. While any cell can produce IFN-I, Plasmacytoid Dendritic Cells (pDCs) make greater quantities and more varieties of these cytokines than any other cell type2. However, following an initial burst of IFN- I, pDCs lose their exceptional IFN-I production capacity and become "exhausted", a phenotype that associates with enhanced susceptibility to secondary infections3-5. Despite this apparent cost for the host, pDC exhaustion is conserved across multiple species and viral infections, but the underlying mechanisms and the potential evolutionary advantages are not well understood. Here we characterize pDC exhaustion and demonstrate that it is associated with a reduced capacity of pDCs to engage both oxidative and glycolytic metabolism. Mechanistically, we identify lactate dehydrogenase B (LDHB) as a novel positive regulator of pDC IFN-I production in mice and humans, show that LDHB deficiency is associated with suppressed IFN-I production, pDC metabolic capacity, and viral control following a viral infection, and demonstrate that preservation of LDHB expression is sufficient to partially restore exhausted pDC function in vitro and in vivo. Furthermore, restoring LDHB in vivo in exhausted pDCs increased IFNAR dependent infection- associated pathology. Therefore, our work identifies a novel and conserved mechanism for balancing immunity and pathology during viral infections, while also providing insight into the highly preserved but previously unexplained phenomenon of pDC exhaustion.

immunology↗

bioRxiv2024

Determination of Absolute Intramolecular Distances in Proteins by Anomalous X-ray Scattering Interferometry

Biomolecular structures are typically determined using frozen or crystalline samples. Measurement of intramolecular distances in solution can provide additional insights into conformational heterogeneity and dynamics of biological macromolecules and their complexes. The established molecular ruler techniques used for this (NMR, FRET, and EPR) are, however, limited in their dynamic range and require model assumptions to determine absolute distance (distributions). Here, we introduce anomalous X-ray scattering interferometry (AXSI) for intramolecular distance measurements in proteins, which are labeled at two sites with small gold nanoparticles of 0.7 nm radius. We apply AXSI to two different cysteine-variants of maltose binding protein in the presence and absence of its ligand maltose and find distances in quantitative agreement with single-molecule FRET experiments. Our study shows that AXSI enables determination of absolute intramolecular distance distributions under virtually arbitrary solution conditions and we anticipate its broad use to characterize protein conformational ensembles and dynamics.

biophysics↗

bioRxiv2024

Rapid and specific detection of single nanoparticles and viruses in microfluidic laminar flow via confocal fluorescence microscopy

Mainstream virus detection relies on the specific amplification of nucleic acids via polymerase chain reaction, a process that is slow and requires extensive laboratory expertise and equipment. Other modalities, such as antigen-based tests, allow much faster virus detection but have reduced sensitivity. In this study, we report the development of a flow virometer for the specific and rapid detection of single nanoparticles based on confocal microscopy. The combination of laminar flow and multiple dyes enable the detection of correlated fluorescence signals, providing information on nanoparticle volumes and specific chemical composition properties, such as viral envelope proteins. We evaluated and validated the assay using fluorescent beads and viruses, including SARS-CoV-2. Additionally, we demonstrate how hydrodynamic focusing enhances the assay sensitivity for detecting clinically-relevant virus loads. Based on our results, we envision the use of this technology for clinically relevant bio-nanoparticles, supported by the implementation of the assay in a portable and user-friendly setup. O_FIG O_LINKSMALLFIG WIDTH=184 HEIGHT=200 SRC="FIGDIR/small/573251v3_ufig1.gif" ALT="Figure 1"> View larger version (97K): org.highwire.dtl.DTLVardef@11781bdorg.highwire.dtl.DTLVardef@46d845org.highwire.dtl.DTLVardef@120d399org.highwire.dtl.DTLVardef@240022_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Single-molecule detection and super-resolution imaging with a portable and adaptable 3D-printed microscopy platform (Brick-MIC)

Over the past decades, single-molecule and super-resolution microscopy have advanced and represent essential tools for life science research. There is,however, a growing gap between the state-of-the-art and what is accessible to biologists, biochemists, medical researchers or labs with financial constraints. To bridge this gap, we introduce Brick-MIC, a versatile and affordable open-source 3D-printed micro-spectroscopy and imaging platform. Brick-MIC enables the integration of various fluorescence imaging techniques with single-molecule resolution within a single platform and exchange between different modalities within minutes. We here present variants of Brick-MIC that facilitate single-molecule fluorescence detection, fluorescence correlation spectroscopy and super-resolution imaging (STORM and PAINT). Detailed descriptions of the hardware and software components, as well as data analysis routines are provided, to allow non-optics specialist to operate their own Brick-MIC with minimal effort and investments. We foresee that our affordable, flexible, and open-source Brick-MIC platform will be a valuable tool for many laboratories worldwide.

biophysics↗

bioRxiv2023

Dissecting Mechanisms of Ligand Binding and Conformational Changes in the Glutamine-Binding Protein

The glutamin-binding protein GlnBP is part of an ATP-binding cassette transporter system in E. coli and uses two well-characterized conformational states, an open ligand-free and a closed-liganded state, to facilitate active amino-acid uptake. Existing literature on its ligand binding mechanism lacked sufficient evidence to univocally assign the kinetic type of binding mechanism for GlnBP: ligand binding prior to conformational change, i.e., an induced fit or the conformational selection, in which the ligand binds the matching conformation from a pre-existing ensemble. Since such mechanistic questions are relevant for our fundamental understanding of how this and other biomacromolecules regulate cellular processes, we here revisit the question for GlnBP. We present a biochemical and biophysical analysis using a combination of calorimetry, single-molecule and surface-plasmon resonance spectroscopy and molecular dynamics simulations. We found that both apo- and holo-GlnBP show no detectable exchange between open and (semi-)closed conformations on timescales between 100 ns and 10 ms and that ligand binding and conformational changes in GlnBP are correlated. A global analysis of our experimental results suggests that the conformational selection model is only compatible with GlnBP for the extreme scenario of very fast conformational exchange between the open and closed states on timescales <100 ns. In contrast all data remains compatible with an induced-fit mechanism, where the ligand binds GlnBP prior to conformational rearrangements. Importantly, our work demonstrates that it is an intricate task to identify the type of kinetic binding mechanism and that this requires not only a sufficient set of data, but also an integrative experimental and theoretical framework to address the question. Based on this concept, we propose that various protein systems, for which so far only insufficient kinetic data are available, should be revisited.

biophysics↗

bioRxiv2023

Labelizer: systematic selection of protein residues for covalent fluorophore labeling

An essential requirement for the use of fluorescent dyes in biomedicine, molecular biology, biochemistry, biophysics and optical imaging is their (covalent) attachment to biomolecules. There is, however, no systematic and automated approach for the selection of suitable labeling sites in macromolecules, which is particular problematic for proteins. Here, we present a general and quantitative strategy to identify optimal residues for protein labeling using a naive Bayes classifier. Based on a literature search and bioinformatics analysis of >100 proteins with ~400 successfully labeled residues, we identified four parameters, which we combined into a labeling score to rank residues for their suitability as a label-site. The utility of our approach for the systematic selection of single residues and of residue pairs for FRET experiments is supported by data from the literature and by new experiments on different proteins. To make the method available to a large community of researchers, we developed a python package called "labelizer", that performs an analysis of a pdb-structure (or structural models), label score calculation, and FRET assay scoring. We further provide a webserver (https://labelizer.bio.lmu.de/) to conveniently apply our approach and to build up a central open-access database of (non-)successfully labeled protein residues to continuously improve and refine the labelizer approach.

biophysics↗

bioRxiv2023

EGFR+ lung adenocarcinomas coopt alveolar macrophage metabolism and function to support EGFR signaling and growth

The limited efficacy of currently approved immunotherapies in EGFR-mutant lung adenocarcinoma (LUAD) underscores the need to better understand mechanisms governing local immunosuppression. Elevated surfactant and GM-CSF secretion from the transformed epithelium induces tumor-associated alveolar macrophages (TA-AM) to proliferate and support tumor growth by rewiring inflammatory functions and lipid metabolism. TA-AM properties are driven by increased GM-CSF--PPAR{gamma} signaling and inhibition of airway GM-CSF or PPAR{gamma} in TA-AMs suppresses cholesterol efflux to tumor cells, which impairs EGFR phosphorylation and restrains LUAD progression. In the absence of TA-AM metabolic support, LUAD cells compensate by increasing cholesterol synthesis, and blocking PPAR{gamma} in TA-AMs simultaneous with statin therapy further suppresses tumor progression and increases T cell effector functions. These results reveal new therapeutic combinations for immunotherapy resistant EGFR-mutant LUADs and demonstrate how such cancer cells can metabolically co-opt TA-AMs through GM-CSF--PPAR{gamma} signaling to provide nutrients that promote oncogenic signaling and growth.

immunology↗

bioRxiv2022

Non-targeted metabolomics identifies erythronate accumulation in cancer cells

Using a non-targeted isotope-assisted metabolomics approach, we identified erythronate as a metabolite that accumulates in several human cancer cell lines. Erythronate has been reported to be a detoxification product derived from off-target glycolytic metabolism. We provide data supporting a possible alternative route to erythronate production involving the dephosphorylation of the pentose phosphate pathway intermediate erythrose-4-phosphate to form erythrose, followed by the oxidation of erythrose by an aldehyde dehydrogenase. Finally, we detected increased erythronate concentrations in tumors relative to adjacent normal tissues from lung cancer patients. These findings suggest the accumulation of erythronate to be an example of metabolic reprogramming in cancer cells, raising the possibility that elevated level of erythronate may serve as a biomarker of certain types of cancer.

biochemistry↗

bioRxiv2022

Inter-organelle crosstalk supports acetyl-coenzyme A homeostasis and lipogenesis under metabolic stress

Proliferating cells rely on acetyl-CoA to support membrane biogenesis and acetylation. Several organelle-specific pathways are available for provision of acetyl-CoA as nutrient availability fluctuates, so understanding how cells maintain acetyl-CoA flux under such stresses is critically important. To this end we applied 13C isotope tracing cell lines deficient in these mitochondrial (ATP-citrate lyase; ACLY-), cytosolic, (acetyl-CoA synthetase (ACSS2-), and peroxisomal (peroxisomal biogenesis factor 5; PEX5-) dependent pathways. ACLY knockout in multiple cell lines reduced fatty acid synthesis and increased reliance on extracellular lipids or acetate. Knockout of both ACLY and ACSS2 (DKO) severely stunted but did not entirely block proliferation, suggesting alternate pathways can support acetyl-CoA homeostasis. Metabolic tracing and PEX5 knockout studies link peroxisomal oxidation of exogenous lipids as a major source of acetyl-CoA for lipogenesis and histone acetylation, highlighting a role for inter-organelle crosstalk in supporting cell survival in response to nutrient fluctuations. TeaserWe quantify how acetyl-CoA metabolism is supported by distinct pathways spanning mitochondria, cytosol, and peroxisomes using comprehensive tracing applied to knockout cells.

bioengineering↗