Biology subjects

Walter, S.

Publications and source records attributed to Walter, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Systematic Analysis of GARP- and EARP-Mediated Trafficking Pathways Using Lysosomal Proteomics

Eukaryotic cells utilize tethering complexes for sorting of proteins within the endo-lysosomal system. The GARP complex and the EARP complex both function in endosomal recycling. Both complexes share three common subunits (VPS51, VPS52, and VPS53), with each relying on one specific subunit: VPS54 for GARP and VPS50 for EARP. Mutations in both complexes are linked to various neurodegenerative disorders. While model cargoes have been described, the cargo spectrum of both complexes remains largely elusive. Here, we combined lysosomal proteomics with knockdowns of GARP and EARP to analyze their cargo spectra. Using the lysosome as a terminal reference point for mis-sorted proteins, we show that GARP depletion causes a loss in lysosomal hydrolases and accumulation of non-lysosomal cargo. EARP depletion results in lysosomal accumulation of secreted proteins. Our findings serve as a reference to study GARP and EARP complexes. We demonstrate that a systematic phenotypic analysis of GARP and EARP mutants is necessary to understand the pathological consequences of their dysfunction.

biochemistry↗

bioRxiv2026

Lipid Droplet Targeting Drives Substrate Access during Very Long Chain Fatty Acid Activation by Fat1

Fatty acids, the most abundant building blocks for lipid synthesis, require conjugation with Coenzyme A (CoA) for their incorporation into membrane lipids. Of those, very long chain fatty acids (VLCFAs), the precursors of yeast ceramides, are particularly hydrophobic. Yet it remains unresolved where and how the sole VLCFA-CoA synthetase Fat1 activates them. Here we show that lipid droplets (LDs) are both the site of free VLCFAs storage, as well as their place of activation. Free VLCFAs preferentially partition into LDs in molecular simulations and in vitro reconstitutions, whereas Fat1 targets the LD surface in cells via a N-terminal amphipathic helix. Structural predictions identify an essential hydrophobic cavity in Fat1 that connects the active site to the membrane, suggesting that VLCFAs directly shuttle from the LD core into the enzymatic pocket. Our model explains how the organelle targeting of a fatty acid activator evolved to match the biophysical properties and consequently the subcellular localization of its highly hydrophobic substrate.

biochemistry↗

bioRxiv2026

Membrane-Promoted J-Aggregation of BODIPY Dimers Enables Spontaneous Blinking in Green and Red Channels for Live-Cell Nanoscopy

BODIPY dyes are widely used in bioimaging, yet their aggregation behavior within the plasma membrane (PM) remains poorly exploited for single-molecule localization microscopy (SMLM). Here, we design a series of green-emitting PM-targeted BODIPY dimers engineered to undergo spontaneous and transient H- and J-aggregation. By tuning the linker length between the fluorophores, we identify dimers that form intramolecular H-aggregates in polar media and emissive J-aggregates ({lambda}em {approx} 535 nm) through membrane-driven intermolecular interactions. In lipid bilayers, all dimers aggregate above a probe/lipid ratio of 1/100, exclusively generating J-aggregates. In live-cell SMLM, the monomeric MB-488 provides high event numbers via diffusion-driven emission, whereas dimers exhibit stable blinking and yield brighter red-shifted J-aggregate events with improved localization precision. Red-channel events localize to specific PM regions, suggesting preferential J-aggregation within distinct membrane microdomains. HaloTag constructs targeted to cell-surface PDGFR further confirm that intramolecular J-aggregation is possible but strongly amplified by the membrane through intermolecular collisions. These results demonstrate that green BODIPYs and their J-aggregates enable robust live SMLM in green and red channels, and reveal the PM as a privileged environment for emissive J-aggregation.

biophysics↗

bioRxiv2026

systematic evaluation and benchmarking of text summarization methods for biomedical literature: From word-frequency methods to language models

The rapid expansion of biomedical literature demands automated summarization tools that reliably condense research articles into concise, accurate summaries. We benchmarked 62 summarization methods, ranging from frequency-based and TextRank extractors to encoder-decoder models (EDMs) and large language models (LLMs), on 1,000 biomedical abstracts from 20 journals across ScienceDirect and Cell Press, using author-written highlights as reference summaries. Models were evaluated with a composite suite of lexical, semantic, and factual metrics, including ROUGE, BLEU, METEOR, embedding-based similarity, and factuality scores. General-purpose models (e.g., Mistral, GPT, Llama) achieved the highest overall performance across lexical and semantic dimensions, outperforming reasoning-oriented (e.g., DeepSeek, Magistral) and domain-specific (e.g., BioGPT, BioMistral) models. Notably, medium-sized models outperformed large-scale models, suggesting an optimal balance between model capacity and efficiency, while classical extractive methods lagged behind neural approaches. These findings provide a systematic reference for selecting biomedical summarization tools and highlight that broad pretraining outperforms narrow domain adaptation.

bioinformatics↗

bioRxiv2025

Reciprocal Regulation Between the SCFFBXO24 Ubiquitin E3 Ligase and FoxP1 Protein

Forkhead Box Protein P1 (FoxP1) is a crucial transcriptional repressor essential for the development of the brain and heart. In adults, FoxP1 protein levels are dysregulated in a variety of disorders, including chronic obstructive pulmonary disease (COPD), atherosclerosis, and heart failure, where they causally contribute to disease pathogenesis. Although independent investigators have reported that FoxP1 protein is ubiquitinated, and E3 ligases have been identified for other FoxP family proteins, the identity of the E3 ligase that controls FoxP1 protein stability has remained unknown. Here, we identify FBXO24, a subunit of the Skp-Cullin-F-box (SCF) ubiquitin E3 ligase complex, as the regulator of FoxP1 ubiquitination and stability. Specifically, FBXO24 regulates K48 and K63 ubiquitination, complexes with, and co-localizes to the nucleus with FoxP1 protein in lung epithelial cells. Depleting FBXO24 reverses the unfolded protein response and cell death triggered by loss of FoxP1 protein in lung epithelium, suggesting a protective role. Additionally, FBXO24 knockout mice exhibit elevated FoxP1 levels in the lung and heart and reduced unfolded protein response activity after short-term cigarette smoke exposure. Intriguingly, we also uncovered bidirectional regulation, whereby FoxP1 protein binds to the FBXO24 promoter to suppress FBXO24 transcription. To our knowledge, this is the first evidence that a substrate for an E3 ligase can also regulate the E3 ligase and, therefore, control levels of other substrates, revealing new regulatory networks. Targeting FBXO24 may offer a therapeutic strategy for COPD, atherosclerosis, and heart failure by stabilizing FoxP1 levels in the heart and lungs and mitigating harmful downstream effects.

molecular biology↗

bioRxiv2025

Determining preclinical safety of Aclarubicin in pediatric malignancies

BackgroundAnthracyclines are among the most effective chemotherapeutic agents used to treat pediatric malignancies. However, their clinical use is limited by dose-dependent toxicities, particularly cardiotoxicity and secondary malignancies. Aclarubicin (Acla) is an anthracycline derivative that induces chromatin damage while sparing DNA, offering potential therapeutic benefit with reduced longterm toxicity. MethodsWe evaluated the anti-tumor efficacy and safety profile of Acla in multiple in vitro pediatric cancer models and in vivo mouse models designed to mimic anthracycline re-treatment following prior doxorubicin (Doxo) exposure. Tumor growth, genotoxic stress, survival, and organ toxicity were assessed. ResultsAcla demonstrated robust anti-tumor activity comparable to Doxo across diverse pediatric in vitro models. Unlike Doxo, Acla treatment did not induce significant genotoxic stress. In vivo, mice receiving Acla after Doxo exposure showed no evidence of cumulative cardiotoxicity or end-organ damage. In contrast, a second course of Doxo led to significant toxic mortality, but was surprisingly not attributable to classic cardiac injury. ConclusionOur study highlights Acla as a promising anthracycline derivative for pediatric cancers, with potent anti-tumor efficacy and a superior safety profile, even following prior anthracycline exposure. These results support continued investigation of chromatin-damaging anthracyclines that can kill pediatric cancer cells without inducing genotoxic stress. In addition, our studies underscore the need to refine preclinical models to better understand both acute and chronic anthracycline toxicities in pediatric and adolescent populations.

cancer biology↗

bioRxiv2024

Approach-Avoidance Bias in Virtual and Real-World Simulations: Insights from a Systematic Review of Experimental Setups

BackgroundApproach and avoidance bias (AAB) describes automatic behavioral tendencies to react toward environmental stimuli regarding their emotional valence. Traditional setups have provided evidence but often lack ecological validity. The study of the AAB in naturalistic contexts has recently increased, revealing significant methodological challenges. This systematic review evaluates the use of virtual reality (VR) and real-world setups to study the AAB, summarizing methodological innovations and challenges. MethodsWe systematically reviewed peer-reviewed articles employing VR and real-world setups to investigate the AAB. We analyzed experimental designs, stimuli, response metrics, and technical aspects to assess their alignment with research objectives and identify limitations. ResultsThis review included 21 studies revealing diverse methodologies, stimulus types, and novel behavioral responses, highlighting significant variability in design strategies and methodological coherence. Several studies used traditional reaction time measures yet varied in their application of VR technology and participant interaction paradigms. Some studies showed discrepancies between simulated and natural bodily actions, while others showcased more integrated approaches that preserved their integrity. Only a minority of studies included control conditions or acquired (neuro)physiological data. ConclusionsVR offers a potential ecological setup for studying the AAB, enabling dynamic and immersive interactions. Our results underscore the importance of establishing a coherent framework for investigating the AAB tendencies using VR. Addressing the foundational challenges of developing baseline principles that guide VR-based designs to study the AAB within naturalistic contexts is essential for advancing the AAB research and application. This will ultimately contribute to more reliable and reproducible experimental paradigms and develop effective interventions that help individuals recognize and change their biases, fostering more balanced behaviors.

neuroscience↗

bioRxiv2024

The Small GTPase Rho5 - Yet Another Player in Yeast Glucose Signaling

The small GTPase Rho5 has been shown to be involved in regulating the Bakers yeast response to stress on the cell wall, high medium osmolarity, and reactive oxygen species. These stress conditions trigger a rapid translocation of Rho5 and its dimeric GDP/GTP exchange factor (GEF) to the mitochondrial surface, which was also observed upon glucose starvation. We here show that rho5 deletions affect carbohydrate metabolism both at the transcriptomic and the proteomic level, in addition to cell wall and mitochondrial composition. Epistasis analyses with deletion mutants in components of the three major yeast glucose signaling pathways indicate a primary role of Rho5 upstream of the Ras2 GTPase in cAMP-mediated protein kinase A signaling. We also observed an inhibitory role of Rho5 on respiratory capacity, which may be explained by its role in mitophagy.

molecular biology↗

bioRxiv2024

Representing Transcription Factor Dimer Binding Sites Using Forked-Position Weight Matrices and Forked-Sequence Logos

Current position weight matrices and sequence logos may not be sufficient for accurately modeling transcription factor binding sites recognized by a mixture of homodimer and heterodimer complexes. To address this issue, we developed forkedTF, an R-library that allows the creation of Forked-Position Weight Matrices (FPWM) and Forked-Sequence Logos (F-Logos), which better capture the heterogeneity of TF binding affinities based on interactions and dimerization with other TFs. Furthermore, we have enhanced the standard PWM format by incorporating additional information on co-factor binding and DNA methylation. Precomputed FPWM and F-Logos are made available in the MethMotif 2024 database, thereby providing ready-to-use resources for analyzing TF binding dynamics. Finally, forkedTF is designed to support the TRANSFAC format, which is compatible with most third-party bioinformatics tools that utilize PWMs. The forkedTF R-library is open source and can be accessed on GitHub at https://github.com/benoukraflab/forkedTF.

bioinformatics↗

bioRxiv2024

DNA virus infections shape transposable elements activity in vitro and in vivo

Transposable elements (TEs) are implicated in a variety of processes including placental and preimplantation development and a variety of human diseases. TEs are known to be activated in the context of some viral infections, but the mechanisms and consequences are not understood. We show strong activation of TEs upon DNA virus infection, in particular the MLT- and THE1-class of LTR-containing retrotransposons as well as a subset of LINE-1-, Alu-elements and HERVs. Mechanistically, two key pathways induce TEs upregulation: inhibition of the KAP1/TRIM28 repressive complex by phosphorylation, and expression of the pioneer transcription factor double-homeobox 4 (DUX4), which is known to be involved in TE-induction during zygotic genome activation in embryonic development. DUX4 is induced by DNA viruses, it binds to TEs upon infection and analysis of genes adjacent to TEs shows pathways that are important for DNA virus infections. Analysis of knockdown, knockout and overexpression data reveal that almost all TEs expressed upon herpesviral infection are regulated by KAP1/TRIM28 and DUX4. Interestingly, analysis of single cell sequencing data from patients with DNA virus-associated cancers showed that in vivo TEs expression strongly correlates with virus infection, indicating a possible role in viral oncogenesis.

genomics↗

bioRxiv2024

The structure of the Orm2-containing serine palmitoyltransferase complex reveals distinct inhibitory potentials of yeast Orm proteins.

The levels of sphingolipids are crucial determinants of neurodegenerative disorders. Therefore, sphingolipid levels have to be tightly regulated. The Orm protein family and ceramides act as inhibitors in the rate-limiting step of sphingolipid biosynthesis - the condensation of L-serine and palmitoyl-CoA. The two yeast isoforms Orm1 and Orm2 form a complex with the serine palmitoyltransferase (SPT). While the sequences of the Orm proteins are highly similar, however, Orm1 and Orm2 are differentially regulated in yeast cells. The mechanistic details of the differential regulation remain elusive. To elucidate the regulatory mechanism, we determined the cryo-electron microscopy structure of the SPT complex containing Orm2. Through in vitro activity assays and a newly developed plasmid coverage assay combined with targeted lipidomics, we demonstrate that the SPT complex with Orm2 exhibits lower activity in comparison to the complex containing Orm1. This collectively suggests a higher inhibitory potential of Orm2, despite the remarkably similar structures of the Orm1- and Orm2 containing complexes. The high conservation of the SPT from yeast to man implies that the three human ORMDL isoforms could also have different regulatory capacities, which might be essential to understanding their role in sphingolipid-mediated neurodegenerative disorders. HighlightsO_LICryo-EM structures of serine palmitoyltransferase with Orm2 from S. cerevisiae C_LIO_LICeramide-mediated inhibition on SPT activity is conserved amongst SPTs C_LIO_LIThe two yeast Orm isoforms have different regulatory capacity C_LI

biochemistry↗

bioRxiv2023

Ventilation does not affect influenza virus transmission efficiency in a ferret playpen setup

Sustained community spread of influenza viruses relies on efficient person-to-person transmission. Current experimental transmission systems do not mimic environmental conditions (e.g., air exchange rates, flow patterns), host behaviors or exposure durations relevant to real-world settings. Therefore, results from these traditional systems may not be representative of influenza virus transmission in humans. To address this pitfall, we developed a modified, more realistic transmission setup and used it to investigate the impact of ventilation rates on transmission in a close-range, play-based scenario. In this setup, four immunologically naive recipient ferrets were exposed to a donor ferret infected with a genetically barcoded 2009 H1N1 virus (H1N1pdm09) for four hours. The ferrets interacted in a shared space that included toys, similar to a child care setting. The transmission efficiency was determined under conditions of low and high ventilation rates; air exchange rates of [~] 1.3 hr-1 and 23 hr-1, respectively. Despite the large difference in ventilation rate, transmission efficiency was the same, 50% in two independent replicate studies. The presence of infectious virus or viral RNA on surfaces and in air throughout the exposure area was similar regardless of ventilation rate. While high viral genetic diversity in donor ferret nasal washes was maintained during infection, recipient ferret nasal washes displayed low diversity, revealing a narrow transmission bottleneck regardless of ventilation rate. Our findings indicate that in exposures characterized by frequent close-range, play-based interactions and the presence of fomites, ventilation does not significantly impact transmission efficiency. SignificanceImproved ventilation in building has the potential to reduce transmission of respiratory viruses, but its effect in different settings is not well understood. We developed a novel system to study influenza virus transmission in the ferret animal model in an environment that mimics a child care center. We demonstrate that increased ventilation is not effective at disrupting transmission in this setting, suggesting that transmission occurs mainly at close-range or via fomites. Multiple interventions are needed to reduce the spread of influenza virus in this type of setting.

microbiology↗

bioRxiv2023

Structure of the yeast ceramide synthase

Ceramides play a pivotal role as essential lipids, serving as foundational components for complex sphingolipids and potent signaling molecules. Ceramides are the products of the N-acylation of a sphingoid base and a CoA-activated fatty acid. This reaction is catalyzed by the enzymes of the evolutionarily conserved ceramide synthase (CerS) family. Yet, the precise structural details and catalytic mechanisms of CerSs have remained elusive. Here, we employed cryo-EM single particle analysis to unravel the structure of the yeast ceramide synthase complex in both an active and a fumonisin B1 inhibited state. Our findings shed light on the complexs architecture, revealing a dimer of Lip1 subunits bound to the two catalytic subunits, Lag1 and Lac1. Each catalytic subunit forms a hydrophobic crevice that is accessible from both the cytosolic site as well as from the intermembrane space of the endoplasmic reticulum (ER). Within this cavity, we identify amino acids forming the active center and a sphingoid base, one of the substrates of the complex. Together, this suggests a pre-loaded state of the CerS. Additionally, the fumonisin B1 bound structure reveals the inhibitory mechanism by blocking the cytosolic acyl-CoA binding site.

biochemistry↗

bioRxiv2023

Structure of the endosomal CORVET tethering complex

Cells depend on their endolysosomal system for nutrient uptake and downregulation of plasma membrane proteins. These processes rely on endosomal maturation, which requires multiple membrane fusion steps. Early endosome fusion is promoted by the Rab5 GTPase and its effector, the hexameric CORVET tethering complex, which is homologous to the lysosomal HOPS. How these related complexes recognize their specific target membranes remains entirely elusive. Here, we solved the structure of CORVET by cryo-electron microscopy and revealed its minimal requirements for membrane tethering. As expected, the core of CORVET and HOPS resembles each other. However, the function-defining subunits show marked structural differences. Notably, we discovered that unlike HOPS, CORVET depends not only on Rab5 but also on phosphatidylinositol-3-phosphate (PI3P) and membrane packaging defects for tethering, implying that an organelle-specific membrane code enables fusion. Our data suggest that both shape and membrane interactions of CORVET and HOPS are conserved in metazoans, thus providing a paradigm how tethering complexes function. One-Sentence SummaryUsing cryo-EM, we solved the structure of the CORVET tethering complex and explained how it promotes endosome fusion.

biochemistry↗

bioRxiv2023

Complex Sphingolipid Profiling and Identification of an Inositol Phosphorylceramide Synthase in Dictyostelium discoideum

Dictyostelium discoideum is a professional phagocyte frequently used as experimental model to study cellular processes underlying the recognition, engulfment and infection course of microbial pathogens. Sphingolipids are abundant components of the plasma membrane that bind cholesterol, control vital membrane properties, participate in signal transmission and serve as adhesion molecules in recognition processes relevant to immunity and infection. While the pathway of sphingolipid biosynthesis has been well characterized in plants, animals and fungi, the identity of sphingolipids produced in D. discoideum, an organism at the crossroads between uni- and multicellular life, is not known. Combining lipidomics with a bioinformatics-based cloning strategy for key sphingolipid biosynthetic enzymes, we show here that D. discoideum produces phosphoinositol-containing sphingolipids with predominantly phytoceramide backbones. Cell-free expression of candidate inositol-phosphorylceramide (IPC) synthases from D. discoideum in defined lipid environments enabled identification of an enzyme that selectively catalyses the transfer of phosphoinositol from phosphatidylinositol onto ceramide. The corresponding IPC synthase, DdIPCS1, is non-homologous to but shares multiple sequence motifs with yeast IPC and human sphingomyelin synthases and localizes to the Golgi apparatus as well as the contractile vacuole of D. discoideum. Collectively, these findings open up important opportunities for exploring a role of sphingolipids in phagocytosis and infection across major evolutionary boundaries.

cell biology↗

bioRxiv2023

Utilizing a nanobody recruitment approach for assessing serine palmitoyltransferase activity in ER sub-compartments of yeast.

Sphingolipids (SP) are one of the three major lipid classes in eukaryotic cells and serve as structural components of the plasma membrane. The rate-limiting step in SP biosynthesis is catalyzed by serine palmitoyltransferase (SPT). In yeast, SPT consists of two catalytic subunits (Lcb1 and Lcb2), a regulatory subunit (Tsc3), negative regulators (Orm1 and Orm2), and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1, collectively known as the SPOTS complex. Regulating SPT activity enables cells to adapt SP metabolism to changing environmental conditions. Therefore, the Orm proteins are phosphorylated by two signaling pathways originating from either the plasma membrane localized target of rapamycin (TOR) complex 2 or the lysosomal/vacuolar TOR complex 1. Moreover, uptake of exogenous serine is necessary for the regulation of SP biosynthesis, which suggests the existence of differentially regulated SPT pools based on their intracellular localization. However, tools for measuring lipid metabolic enzyme activity in different cellular compartments are currently not available. We have developed a nanobody recruitment system that enables the re-localization of the SPOTS complex to the nuclear or peripheral ER. By combining this system with sphingolipid flux analysis, we have identified two distinct active SPT pools in cells. Our method thus serves as a new and versatile tool to measure lipid metabolism with sub-cellular resolution.

biochemistry↗

bioRxiv2023

Structure of the ceramide-bound SPOTS complex

Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyl-transferase (SPT) catalyzes the rate limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.

biochemistry↗

bioRxiv2022

Streetlights affect moth orientation beyond flight-to-light behaviour

One of the most dramatic changes occurring on our planet in recent decades is the ever-increasing extensive use of artificial light at night, which drastically altered the environment nocturnal animals are adapted to 1,2. One nocturnal species group experiencing marked declines are moths, which are not only of great importance for species conservation, but also for their key role in food webs and in ecosystem services such as nocturnal plant pollination 3,4. Light pollution has been identified as a driver in the dramatic insect decline of the past years 5-7, yet little is known about its impact on natural insect orientation behaviour. Using harmonic radar tracking, we show that the orientation of several species of moths is significantly affected by streetlights, although only 4 % of individuals showed flight-to-light behaviour. We reveal a species-specific barrier effect of streetlights on lappet moths whenever the moon was not available as a natural celestial cue. Furthermore, streetlights increased the tortuosity of flight trajectories for both hawk moths and lappet moths. Our results provide the first spatially resolved experimental evidence for the fragmentation of landscapes by streetlights and demonstrate that light pollution affects movement patterns of moths beyond previously assumed extend, potentially affecting their reproductive success and hampering a vital ecosystem service.

animal behavior and cognition↗