Biology subjects

Koehler, M.

Publications and source records attributed to Koehler, M..

12 recordsLinked to original sources

bioRxiv2026

Membrane controlled Mechanoregulation in PIEZO1 Interactions

PIEZO channels are mechanosensitive membrane proteins whose activation is governed by the surrounding lipid environment. However, the direct mechanistic contribution of native membrane composition to the molecular interactions remains unclear. In this study, a systematic comparison is made between PIEZO1 reconstituted in detergent micelles and in cell membrane-derived nanodiscs, which preserve the native lipid composition. Initial characterization employing a combination of atomic force microscopy and coarse-grained molecular dynamics simulations unveils distinct physical signatures of PIEZO1 in these two environments. Single-molecule force spectroscopy measurements demonstrate that interaction between the extracellular domain of PIEZO1 and a specific antibody exhibits unique mechanical responses strongly influenced by the surrounding membrane. In nanodiscs, PIEZO1 exhibits reversible, elastic-like behavior with preserved structural integrity and consistent adhesion forces even when modulated by Yoda1 (agonist) and Dooku1 (antagonist). Conversely, micelles induce a plastic response with altered mechanosensitivity and functional stability. Based on these findings, we propose a possible membrane-mediated force transmission pathway and quantify a simplified interaction energy landscape. Collectively, our findings offer the initial direct evidence of how the native lipid environment mechanistically governs PIEZO1 interactions, establishing native membranes as critical determinants for mechanotransduction.

biophysics↗

bioRxiv2025

Microclimatic heterogeneity is associated with forest structural complexity and biodiversity

Forest microclimates, their dependence on forest structure, and their impact on biodiversity are crucial for future forest management under climate change. However, standard approaches for measuring forest microclimates do not capture within-plot heterogeneity, which, according to the habitat heterogeneity hypothesis, is a key driver of local biodiversity. We quantified horizontal and vertical microclimatic heterogeneity within 30 broad-leaved forest plots in Central Europe using a three-dimensional design with high spatial resolution. Moreover, we examined whether microclimatic heterogeneity differs among silvicultural treatments and whether it can be predicted using forest structure indices derived from laser scanning. Additionally, we explored the relationship between microclimatic heterogeneity and biodiversity. In the understory of canopy gaps, warm and cold habitats co-existed in close proximity, leading to a high horizontal microclimatic heterogeneity. In closed stands with high structural complexity, we found steep gradients of increasing temperature and vapor pressure deficit from the ground to the canopy during mid-day. Canopy cover and forest structural complexity were strong indicators of microclimatic heterogeneity. We found positive relationships between herb layer temperature heterogeneity and the diversity of plants, Hymenoptera, and Diptera. Our results demonstrate that single-point measurements fail to capture the substantial microclimatic heterogeneity within plots, potentially misrepresenting the conditions experienced by forest species. However, laser scanning provides reliable indicators for within- plot microclimatic heterogeneity. With canopy gaps featuring high horizontal microclimatic heterogeneity and promoting the biodiversity of several taxonomic groups, we argue that managing forests for maximized temperature buffering should not be the only strategy to conserve forest biodiversity. HighlightsO_LIHigh small-scale horizontal microclimatic heterogeneity in canopy gaps C_LIO_LISteep vertical microclimatic gradients in closed-canopy forests C_LIO_LICanopy cover and structural complexity: indicators for microclimatic heterogeneity C_LIO_LIPositive relationship between herb layer temperature heterogeneity and biodiversity C_LI

ecology↗

bioRxiv2025

A selective inhibitor of oncogenic JNK signalling perturbs metastatic outgrowth of triple-negative breast cancer through metabolic blockade

Although c-Jun N-terminal Kinase (JNK) represents an attractive anti-cancer target, its pleiotropic functionality limits the use of direct JNK inhibitors. Here, we identify a distinct subcellular pattern of JNK activity as a therapeutic vulnerability in breast cancer, where cytoplasmic JNK activity predicts poor survival outcomes, is elevated in triple-negative breast cancers (TNBC) and is essential for metastatic outgrowth. Mechanistic analyses reveal cytoplasmic JNK acts through multiple mechanisms, with downstream targets involved in cellular metabolism and cytoskeletal regulation. On this basis, we leveraged actin-based phenotypic drug-screening and identified K12, an indirect but selective inhibitor of cytoplasmic JNK that blocks TNBC metastatic outgrowth in vivo. We reveal that K12 inhibits glutaminase-1 and the pyruvate dehydrogenase complex, and that this poly-pharmacology overcomes pyruvate anaplerosis, a known resistance mechanism of existing glutaminase inhibitors. These findings demonstrate the potential of selectively targeting the oncogenic function of JNK, offering new treatment options for early-stage metastatic TNBC.

cancer biology↗

bioRxiv2024

Glioma-associated tertiary lymphoid structures are sites of lymphocyte clonal expansion and plasma cell formation

Adult-type diffuse gliomas, the most common primary brain tumors, pose significant clinical challenges due to limited treatment options, restricted anti-tumor immune response and dismal patient prognosis. In this study, we elucidate the immunological function and clinical relevance of intra-tumoral tertiary lymphoid structures (TLS) in adaptive anti-glioma immunity. We conducted a comprehensive, unbiased analysis of lymphoid aggregation in 642 gliomas using a multi-modal approach that combines RNA sequencing with spatial transcriptome and proteome profiling. Our findings reveal that TLS are present in 15% of tumors and correlate with improved overall survival. Gliomas with TLS exhibit a remodeled perivascular space, marked by transcriptional upregulation and spatial redistribution of collagens associated with barrier functions. Furthermore, we demonstrate that TLS maturation into sites of dynamic adaptive immune responses, characterized by clonal T and B cell expansion and IgA+ and IgG+ plasma-cell formation, is driven by efficient early T cell recruitment to the perivascular space.

immunology↗

bioRxiv2023

Quantitative Impact of Coil Misalignment and Misplacement in Transcranial Magnetic Stimulation

IntroductionTargeting in transcranial magnetic stimulation (TMS) involves the accurate placement and positioning of the stimulation coil on the head of a subject or patient. In clinical and research applications, this placement is even done manually and/or with fixed coil holders that do not compensate for motion and drift of the head. The placement involves six degrees of freedom (DOF; three position DOF: 1x contact and 2x head location; three rotational DOF: 2x alignment and 1x electric field direction/orientation), which challenge operators. This procedure is--even with an experienced user--prone to positioning errors, which can result in low treatment efficacy or high stimulation strength due to overestimating the resting motor threshold (RMT). Whereas the position and field orientation are at least widely appreciated, the coil-head alignment and its impact are often not even known. Errors involve constant errors, drift (both leading to bias and inter-individual variability), and particularly fluctuations (causing intra-individual variability). ObjectiveWe demonstrate the impact of positioning error on cortical field strength to get a better understanding of the importance of accurate positioning and compare as well as quantify the impact of position vs. alignment errors. MethodsWe simulated the impact in a realistic head anatomy to quantify various levels of position errors and misalignment, rolling-off the coil from the target. ResultsPosition and alignment errors shift the focus of the electric field and reduce the electric field in the actual target. A misalignment of 10{degrees} can exceed the loss of stimulation strength in the target associated with a shift of 10 mm, corresponding to threshold stimulation leading to no detectable electromyographic response anymore. Misalignment in the direction of the handle (pitch), with which many operators appear to struggle most, reduces the field in the actual target faster than left-right roll. ConclusionThis work highlights the importance of the coil-head alignment for intra- and interindividual variability.

neuroscience↗

bioRxiv2022

The cohesin subunit RAD21.2 functions as a recombination silencer of ribosomal DNA arrays

Introductory ParagraphIn many species, including Arabidopsis, heterochromatin often comprises repetitive DNA elements, such as arrays of ribosomal DNA (rDNA). Repetitive regions pose a risk in meiosis since recombination between them can lead to gross genomic rearrangements. However, meiotic recombination at rDNA arrays and other heterochromatic repeat regions is blocked by not well understood mechanisms. Here, we have identified RAD21.2, an -kleisin subunit of cohesin, as a repressor of meiotic recombination at the rDNA regions in Arabidopsis. We show that RAD21.2 co-localizes with heterochromatic factors and is specifically enriched at rDNA repeats, which are devoid of the meiosis specific -kleisin REC8, needed for recombination. Knocking down RAD21.2, we find that REC8 moves into the nucleolus organizing regions (NORs), where we see an increase of RAD51 recombinase foci numbers. Concomitantly, we find extensive rearrangements of the NORs and the offspring of these plants have large variation in rDNA copy numbers demonstrating that RAD21.2 is necessary for transgenerational genome stability. One-Sentence SummaryThe cohesin component RAD21.2 represses meiotic recombination and by that contributes to genome stability over generations.

cell biology↗

bioRxiv2021

The human neuronal receptor NgR1 bridges reovirus capsid proteins to initiate infection

Human Nogo-66 receptor 1 (NgR1) is a receptor for mammalian orthoreoviruses (reoviruses), but the mechanism of virus-receptor engagement is unknown. NgR1 binds a variety of structurally dissimilar ligands in the adult central nervous system (CNS) to inhibit axon outgrowth. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron regrowth, making NgR1 an important therapeutic target for diverse conditions such as spinal crush injuries and Alzheimer disease. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that NgR1 sequences in a central concave region of the molecule establish a bridge between two copies of the viral capsid protein, {sigma}3. This unusual binding interface produces high-avidity interactions between virus and receptor and likely primes early entry steps. NgR1 sequences engaged by reovirus also are required for NgR1 binding to ligands expressed by neurons and oligodendrocytes. These studies redefine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

microbiology↗

bioRxiv2020

Hallmarks of Alpha- and Betacoronavirus non-structural protein 7+8 complexes

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses with SARS-CoV-2 causing a pandemic in 2020. Coronaviral non-structural proteins (nsp) built up the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complex has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on a native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations mainly heterotetramers are observed for SARS-CoV-2 nsp7+8. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model is proposed.

biophysics↗

bioRxiv2020

Reovirus σ1 conformational flexibility modulates the efficiency of host cell attachment

Reovirus attachment protein {sigma}1 is a trimeric molecule containing tail, body, and head domains. During infection, {sigma}1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows {sigma}1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in {sigma}1 from a compact form on virions to an extended form on ISVPs. To determine the importance of {sigma}1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can crosslink {sigma}1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that crosslink the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride. Together, these data suggest that {sigma}1 conformational flexibility modulates the efficiency of reovirus host cell attachment. IMPORTANCENonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, {sigma}1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that crosslink {sigma}1 monomers in non-reducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of {sigma}1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that crosslinking the {sigma}1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that {sigma}1 conformational rearrangements mediate the efficiency of reovirus host cell attachment.

microbiology↗

bioRxiv2020

Alterations of multiple alveolar macrophage states in chronic obstructive pulmonary disease

Despite the epidemics of chronic obstructive pulmonary disease (COPD), the cellular and molecular mechanisms of this disease are far from being understood. Here, we characterize and classify the cellular composition within the alveolar space and peripheral blood of COPD patients and control donors using a clinically applicable single-cell RNA-seq technology corroborated by advanced computational approaches for: machine learning-based cell-type classification, identification of differentially expressed genes, prediction of metabolic changes, and modeling of cellular trajectories within a patient cohort. These high-resolution approaches revealed: massive transcriptional plasticity of macrophages in the alveolar space with increased levels of invading and proliferating cells, loss of MHC expression, reduced cellular motility, altered lipid metabolism, and a metabolic shift reminiscent of mitochondrial dysfunction in COPD patients. Collectively, single-cell omics of multi-tissue samples was used to build the first cellular and molecular framework for COPD pathophysiology as a prerequisite to develop molecular biomarkers and causal therapies against this deadly disease.

systems biology↗

bioRxiv2020

High-affinity agonist binding to C5aR results from a cooperative two-site binding mechanism

A current challenge in the field of life sciences is to decipher, in their native environment, the functional activation of cell surface receptors upon binding of complex ligands. Lack of suitable nanoscopic methods has hampered our ability to meet this challenge in an experimental manner. Here, we use for the first time the interplay between atomic force microscopy, steered molecular dynamics and functional assays to elucidate the complex ligand-binding mechanism of C5a with the human G protein-coupled C5a receptor (C5aR). We have identified two independent binding sites acting in concert where the N-terminal C5aR serves as kinetic trap and the transmembrane domain as functional site. Our results corroborate the two-site binding model and clearly identify a cooperative effect between two binding sites within the C5aR. We anticipate that our methodology could be used for development and design of new therapeutic agents to negatively modulate C5aR activity.

biochemistry↗

bioRxiv2019

A global screen for assembly state changes of the mitotic proteome by SEC-SWATH-MS

Living systems integrate biochemical reactions that determine the functional state of each cell. Reactions are primarily mediated by proteins that have in systematic studies been treated as independent entities, disregarding their higher level organization into complexes which affects their activity and/or function and is thus of great interest for biological research. Here, we describe the implementation of an integrated technique to quantify cell state-specific changes in the physical arrangement of protein complexes, concurrently for thousands of proteins and hundreds of complexes. Applying this technique for comparison of human cells in interphase and mitosis, we provide a systematic overview of mitotic proteome reorganization. The results recall key hallmarks of mitotic complex remodeling and discover new events, such as a new model of nuclear pore complex disassembly, validated by orthogonal methods. To support the interpretation of quantitative SEC-SWATH-MS datasets, we extend the software CCprofiler and provide an interactive exploration tool, SECexplorer-cc.\n\nHighlightsO_LIQuantification of proteome assembly state changes using SEC-SWATH-MS\nC_LIO_LISystems-wide analysis of assembly state changes in the mitotic proteome\nC_LIO_LIDiscovery and validation of a novel mitotic disassembly intermediate of the nuclear pore complex\nC_LIO_LIHigher sensitivity and information content compared to thermostability-based approaches for global measurement of proteome states\nC_LIO_LISECexplorer, an online platform to browse results and investigate proteins newly implicated in cell division\nC_LI

systems biology↗